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Applied Biosystems Prism 377 User Manual

1. Save As Default Cancel Save Copy in Save the changes by clicking To Save use the changes as a one time override for the current run Save As Default save the changes as the new defaults in the existing module Note Rather than permanently modifying existing modules we recommend using the Save Copy In command to create a new module This ensures you will always have all the modules designed for use with your instrument Save Copy In create a new module Enter a name for the new module in the dialog box displayed and then click Save Close the run sheet Unused Modules We recommend moving modules that are not used to a folder named Unused Modules This will limit the number of modules displayed in the module pop up menus on the run sheet 9 46 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com List of Modules The table below lists all the modules supplied with ABI PRISM 377 Data Collection Software version 2 1 To keep the number of modules displayed in the pop up menus on the run sheet to a minimum we recommend moving all unused module files to a folder named Unused Modules on the Macintosh hard drive ABI PRISM 377 Data Collection Software version 2 1 module files Standard Modules Chiller Modules N A N A
2. Proceed to Preparing a Run Sheet on page 3 28 to finish setting up the software Instrument Operation 3 27 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Preparing a Run Sheet Save Time by If the same type of run is performed repeatedly on the same instrument you can Setting Run reduce the time spent setting up run sheets by setting sequencing or GeneScan run Sheet Preferences sheet default preferences Setting run sheet preferences means changing the default value of certain fields on the run sheet template to the values used most often The following fields can be set as preferences Operator Lanes Well to read distance Prerun and run modules Auto print Analysis parameters GeneScan run sheets only Gel s matrix file GeneScan run sheets only gt gt gt gt gt o Size standard GeneScan run sheets only Once these preferences are set the preferred values appear automatically on each new run sheet Preferences can be changed as often as necessary either by setting new preference values or by manually selecting new values on the run sheet Instructions for setting run sheet preferences are located in Chapter 5 Setting Preferences Open a New To open a new run sheet Run Sheet Step Action 1 If the run window used Then for the plate check is 2 still open click on that window to make it th
3. Gel Cassette and Pouring Fixtures 604297 Gel Cassette 401991 Gel Pouring Fixture Kit contains top and bottom fixtures clamps and syringe 401969 Top Pouring Fixture 604014 Bottom Pouring Fixture Buffer Chambers and Gasket Kits 4304406 Upper Buffer Chamber 604078 Upper Buffer Chamber obsolete gasket kit available as P N 604524 603875 Lower Buffer Chamber 4304409 Gasket Replacement Kit for upper buffer chamber P N 4304406 604524 Gasket Replacement Kit for upper buffer chamber P N 604078 Parts and Accessories C 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Electrophoresis Cable and Electrode Assemblies 603822 Upper Buffer Chamber Electrode Assembly 603823 Lower Buffer Chamber Electrode Assembly Front Heat Transfer Plate 603833 Front 36 cm Well to Read Heat Transfer Plate C 4 Parts and Accessories Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Two Pitch Eight Channel Loader Suppliers Introduction For your convenience the following tables provide information on suppliers of two pitch eight channel loaders IMPORTANT Contact the companies listed for availability pricing and technical information regarding these products Supplie
4. Cancel ox Sample Sheet Run The default naming convention for sample sheets run folders and run sheets is Folder and Run shown in the File Names Preferences window above For example a sample sheet Sheet Names created at 3 39 PM on June 30 1998 would have a default name of Sample Sheet 1 30 98 3 39 PM The date and time are added as a suffix Folder and file names can be any text string or nothing at all You can also choose not to display the date and time Sample File Names By default Sample File names have a prefix and a suffix Choices for the prefix are lane number or sample name Choices for the suffix are Nothing lt none gt The current date and time lt date gt A serial number reset to zero at the start of each run lt serial number gt A serial number that increments across runs lt global serial number gt gt gt gt The sample name from the file s sample sheet lt sample name gt IMPORTANT Limit the number of characters in sample file names to 27 or less Do not use colons slashes or symbols in sample names 5 8 Setting Preferences Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Changing Folder To change folder and file name preferences and File Name Preferences Step Action 1 If the Preferences dialog box Then is not open open the Windows pull down menu select Preferences and then sel
5. Non optimal thermal cycling parameters Between the denaturation and annealing stages add a 2 minute down ramp time to thermal cycling profile For multiplex PCR a short down ramp time is not necessarily optimal 4 16 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Resolution Troubleshooting Guide Observation Possible Causes Recommended Actions Fuzzy or smeared bands in electropherogram Dirty gel plates Clean plates with Alconox cleaner and a soft cloth then rinse thoroughly with distilled deionized water Excess urea in wells before loading Flush wells immediately before loading Incorrect TBE buffer formulation Remake buffer carefully following protocol Worn or damaged spacers causing variable gel thickness Replace damaged spacers Poor resolution throughout size range Poor quality or old reagents Remake 10X TBE and gel solution stock using fresh reagents from a reliable source IMPORTANT Urea must be ultrapure Formamide degraded to formic acid and formate ions denaturing applications only Use freshly deionized formamide Deionization procedure in Appendix A Note Formamide pH should be between 7 and 9 Small bubble between load and read region Cast gel as described in protocol Note Make sure plates are lint free before pouring gel Incomplete strand
6. gt gt e e e Oo Primer test Two choices are available for viewing DNA sequences text or map format The Map page shows a graphical representation of the sequence and allows viewing of all primers found in relation to template DNA All sequence annotations are shown including restriction endonuclease sites exon intron junction sites and amino acid translations Primer information primer pairs when applicable is displayed in a primer table page The table displays all relevant information according to your preference You decide how the information display is sorted Primer Express software is fully compatible with our AutoAssembler and Sequence Navigator software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The GeneAssist Sequence Analysis System AutoAssembler Sequence Assembly Software GeneAssist is a multi user system used to edit assemble and analyze DNA and protein sequence data The GeneAssist programs are designed to run on the Macintosh platform either independently or interactively with a UNIX server They provide a robust set of tools for sequence analysis and allow you to access and store data in a common file format GeneAssist has three Macintosh programs Factura cleans up sequence data for analysis and alignment It identifies specified vector and ambiguity ranges restriction sites and a specified confidence range It also iden
7. GeneScan Sample Sheet Project Name Color Std Pres Sample Info Description of Fields The information specified on GeneScan sample sheets is described below on the Sample Sheet Name of Field Description Sample Name Name assigned to a sample IMPORTANT Each sample must have a unique name Limit sample names to 27 characters including the default characters Do not use colons slashes or symbols in sample names Project Name Used to identify data transferred to BioLIMS software If a project name is not specified the data transferred to BioLIMS is identified for BioLIMS by the gel file name only software version 2 0 i and up only Project names must be entered in the Project Info Preference prior to preparing a sample sheet Refer to Project Information Preferences in Chapter 5 for instructions and more information Color and Pres Used to indicate the dye colors run in each lane on the gel Required for data to be analyzed automatically See About Automatic Data Analysis on page 9 38 for more information Std Used to indicate the color of the size standard Required for data to be analyzed automatically See About Automatic Data Analysis on page 9 38 for more information Sample Info Additional sample identification information used by ABI PRISM Genotyper DNA Fragment Analysis Software Comments Any additional information 9 20 Data Collection
8. PreRun module Prerun is resumed Samples are electrophoresed into the gel without data collection for approximately two minutes PreRun module Pauses the prerun Wells flushed again and remainder of samples loaded into the even numbered lanes Gel temperature is maintained N A Terminates the prerun Run module Starts the run electrophoresis and data collection 9 40 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Using Commands The Instrument menu contains commands that perform the same functions as the run from the Instrument sheet buttons Menu Windou Start Plate Check Start PreRun Start Run Pause Resume Cancel Run The functions of the commands on the Instrument menu are summarized below To Select this command from the Instrument menu Start a plate check Start Plate Check Start a prerun Start PreRun run Start a run Start Run Temporarily halt a prerun or run so Pause it can be resumed later Resume a prerun or run after ithas Resume been paused Terminate a plate check prerun or Cancel Run Data Collection Software 9 41 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Modules Overview Modules are software files that contain the various settings electrophore
9. www appliedbiosystems com AR Bes ys D Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 08 2001 Part Number 4307164B an Applera business Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Troubleshooting Chapter Contents In this Chapter This chapter contains information you can use to troubleshoot and solve many of the problems that can occur when using this system Quick reference tables are listed on pages 4 3 through 4 19 The remaining portion of the chapter contains procedures referenced in the tables and provides more information on the data collection software and firmware Topic See Page For More Troubleshooting Information 4 2 Gel and Gel Image Troubleshooting Guide 4 3 Instrument Troubleshooting Guide 4 6 Data Collection Software Troubleshooting Guide 4 10 Signal Troubleshooting Guide 4 11 Amplification Troubleshooting Guide 4 16 Resolution Troubleshooting Guide 4 17 Sample Mobility Troubleshooting Guide 4 19 About Troubleshooting Software 4 20 About Troubleshooting Firmware 4 21 Performing a Single Reset 4 22 Performing a Total Reset 4 22 Performing a Cold Boot 4 23 Locating the Firmware Image File 4 24
10. Conditions Frequent monitoring of the water level is required when the external bath is used during standard runs For long periods of use disconnect the external water bath reinstall the water bottle and tubes and use the standard run modules rather than the chiller modules At system installation the software is typically set up so that the standard modules are the ones displayed in the pop up menus on run sheets Chiller modules are stored in a folder called Chiller Modules You must either move the chiller modules to the Modules folder or change the Folder Locations Preference for Modules to the Chiller Modules folder for the software to access the chiller modules Refer to Installing Chiller Modules on page 9 44 in Chapter 9 Data Collection Software and Folder Location Preferences in Chapter 5 Setting Preferences for instructions on how to access standard and chiller modules B 4 Subambient Temperature Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Installing the External Water Bath Positioning the To position the instrument and external water bath Machines Step Action 1 Position the ABI PRISM 377 so the rear of the instrument can be accessed easily 2 Facing the ABI PRISM 377 position the water bath on the right side of the instrument and at the same height Connect the instrument and water bath to an e
11. Contamination of buffer with fluorescent species e g laboratory pen used to label lanes gel plates or comb Do not mark gel plates or comb Lower buffer chamber not clean i e contaminated with fluorescent species Clean and replenish buffer in lower buffer chamber Tilted or deformed bands Small bubble in lane Cast gel as described in protocol Make sure plates are lint free before pouring gel Gel not clamped properly Use clamps of equal tension along the side of the gel The clamps must be placed over the spacers only IMPORTANT of the gel Do not clamp the bottom Dirty plates Clean plates using one of these methods Soak the plates overnight in a 5 solution of Multiterge detergent VWR Scientific P N 34171 010 Use a3 M HCI wash page 4 31 Use an alcoholic KOH wash page 4 30 Lanes appear as smears Impure or degraded TEMED or APS Use fresh reagents Samples are overloaded Follow loading procedure in your protocol or in Chapter 3 Electrophoresis failure due to buffer leak Make sure that the plates are clamped correctly and that the upper buffer chamber gasket makes a proper seal Do not spill buffer behind the upper buffer chamber Wicking can occur Gel image contains vertical red streaks red rain near end of run top of gel image Gel destruction in read region Wrap bottom of gel to prevent drying Run
12. Folder Location and Folder Name Description Settings Folder Macintosh HD System folder ABI Folder Typically set as the ABI Folder The choices in other Preferences dialog boxes and in Run windows use the information contained in this folder for their pop up menus e g dye set primer files instrument matrix files IMPORTANT To automatically analyze data after a run using the sequencing analysis software the Settings folder must be designated as the ABI folder GeneScan Analysis Parameters Macintosh HD GeneScan Analysis XX GS Parameters Folder Contains the parameters used to analyze GeneScan run data and is located in the GeneScan Analysis software folder GeneScan Size Standard Foldert Macintosh HD GeneScan Analysis X X GS Standards Folder Contains the size standard definitions used to analyze GeneScan run data and is located in the GeneScan Analysis software folder IMPORTANT Locations and folder names listed are those at the time of system installation We strongly recommend not changing folder names and locations Displayed in Folder Locations dialog box for GeneScan users only To set the Folder Location preferences Step Action 1 If the Preferences dialog box Then is not open open the Window menu select Preferences and then select Folder Locations is open open the Page pop up menu and select Folder Locations
13. Making Matrix Files for GeneScan ABI PRISM GeneScan Reference Guide ABI 373 and ABI PRISM 377 DNA Sequencers P N 4303188 Lanes Number of lanes in the gel Run Mode This field is displayed for ABI PRISM 377 XL or 96 lane upgrade XL or 96 lane upgrade instruments only It is used to designate the scan mode 96 Lane instruments only Scan XL Scan or Full Scan Operator Operator s name Sample Number These fields are filled in automatically when the Sample Sheet is Sample Name selected Sample File Name Matrix File The same as the Gel s Matrix File listed above Required only if data is to be analyzed automatically at the end of the run Auto Analyze When selected data is automatically transferred to GeneScan analysis software and analyzed at the end of a run See also About Automatic Data Analysis on page 9 38 Analysis Parameters A set of analysis parameters predefined in GeneScan analysis software Selecting analysis parameters is required only if the Auto Analyze feature is selected Otherwise this field can be left blank See also About Automatic Data Analysis on page 9 38 Size Standard A file that contains information on the size standard run with the samples Selecting a size standard is required only if the Auto Analyze feature is selected Otherwise this field can be left blank See also About Automatic Data Analysis on page 9 38 Auto Print When selected
14. Matrix Standard Standard File C Sample name Copy 1 Ax Sample name Copy 2 G Sample name Copy 3 Tesi Sample name Copy 4 For Filter Set E instrument files Follow the procedure Making an Instrument File for Virtual Filter Set E from Matrix Standards on page 7 10 Whenever the protocol indicates a specific matrix standard to be used follow the table below Matrix Standard Standard File dR110 Sample name Copy 1 dR6G Sample name Copy 2 dTAMRA Sample name Copy 3 dROX Sample name Copy 4 7 18 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To create an instrument file from a sample file continued Step Action 4 Apply the newly made matrix back to the original sample file There should be defined peaks and a flat baseline If the baseline is not flat or if there are dips or pull up peaks then the instrument file is wrong and should not be used Storing and Backing Up the Instrument File Introduction Valid instrument files must be placed in the ABI Folder in the System Folder New instrument files should also be backed up Obsolete instrument files should be deleted or archived Follow the steps in the table below after creating and verifying a new instrument file Storing and Backing To properly store and backup instrument files Up
15. Quit File menu commands The File menu contains commands for Creating opening closing and saving files Importing and exporting data to other file types Printing Quitting the application Keyboard shortcut commands standard print dialog box For more Command Description detail see Close Save Standard Macintosh commands Macintosh Save As manual Page Setup Print Quit New Opens run and sample sheets Use New to create a Open new file Use Open to open an existing file Save a Copy In Use to make an extra copy of an existing file for backup or modification The copy can be renamed and stored in _ a different location from the original Import Imports text from files such as those generated by a page 9 27 database into a sample sheet Export Exports data from a sample sheet in tab delimited text page 9 27 format for database applications Print One Prints one copy of the active window Bypasses the 9 10 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Edit Menu Instrument Select All Fill Down Set Scale Show Clipboard Edit menu commands The Edit menu contains Standard editing and selecting commands Spreadsheet editing commands Command for changing the scale in the Electrophoresis History window Command Description Undo
16. b Watch the scan lines during the PreRun Sometimes contaminants migrate out of the gel during the PreRun and all the lanes can then be used Once the gel has reached run temperature click Pause to temporarily halt the prerun IMPORTANT Pause the prerun do not cancel it When paused the pump and heater remain on thereby maintaining the gel temperature 3 36 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Denature the To denature the samples Samples Step Action 1 Finish preparing the samples and bring them up to the final volume as directed in the protocol you are using 2 Load the samples and any other reagents as directed by the protocol into reaction tubes 3 Cap each tube and vortex for 3 5 seconds Spin down the contents of the tubes and load them onto a thermal cycler or heat block Denature the samples as directed by the protocol Remove the samples from the thermal cycler or heat block and immediately place them on ice until ready to load Load the Samples WARNING Ergonomic Hazard Performing loading activities may increase risk of developing the following cumulative trauma disorders repetitive motion or repetitive strain injuries which include but are not limited to tendinitis tenosynovitis epicondylitis strains and or sprains To reduce the risk of experiencing these types o
17. Data Collection Software Sample Sheet and Enter Sample Names Step 1 2 3 4 If matrix standard samples are being run enter a name for each matrix sample To help ensure a robust matrix is produced we strongly recommend you follow these guidelines Leave at least one empty lane between non matrix standard samples and matrix standard samples Leave one empty lane between each matrix standard sample v Lane number on the gel Empty lanes between non matrix and matrix standard samples and between each matrix standard sample 3 20 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Select a Different dye set primer files can be used for the same run as long as the virtual filter DyeSet Primer File set is the same for all samples Dye set primer file names for Rhodamine Terminators are similar to those for BigDye Terminators and can easily be mistaken for one another If the wrong file is selected base spacing in the data will not be noticeably affected C and T bases will be miscalled If you are not sure which file to select refer to the chemistry kit protocol and to DyeSet Primer Files in Chapter 9 To select a dye set primer file Step Action 1 Open the DyeSet Primer pop up menu and select the appropriate file for the first sample You must select a file if you want the data to be analyzed auto
18. Procedure To perform a total reset Step Action 1 Using the eraser end of a pencil or similar object press the red reset button on the back of the instrument twice in rapid succession Quit the data collection software program Relaunch the data collection software program The firmware is automatically downloaded to the instrument This will take 60 90 seconds Firmware Image Sending Firmware Image to Instrument Note If the following dialog box is displayed see Locating the Firmware Image File on page 4 24 amp Firmware Image Y D ABI Prism 377 Firmware 2 Where Is ABI Prism 377 Firmware 4 In response to this prompt quit and then relaunch the data collection software program again Quit application and relaunch for this A action to take effect 4 22 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Step Action Check the CCD pixel position value as follows a Open the Window menu and select Manual Control b Open the Fxn Name menu and select CCD Pixel Position Value The value is displayed in the Value box If it matches the value on the instrument it is ok If it does not match correct the value See CCD Pixel Position Value on page 4 25 for more information Performing a Cold Boot Overview If the computer and instrument are
19. c Use the index number when requesting documents following the procedures below by phone for fax a delivery From the U S or Canada call 1 800 487 6809 or from outside the U S and Canada call 1 858 712 0317 b Follow the voice instructions to order the documents you want Note There is a limit of five documents per request About This Instrument 1 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To order documents Then through the Internet for fax or e mail delivery a Access the Applied Biosystems Technical Support Web site at http Awww appliedbiosystems com techsupp b Under Resource Libraries click the type of document you want c Enter or select the requested information in the displayed form then click Search d In the displayed search results select a check box for the method of delivery for each document that matches your criteria then click Deliver Selected Documents Now or click the PDF icon for the document to download it immediately e Fill in the information form if you have not previously done so then click Deliver Selected Documents Now to submit your order Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for e mail delivery 1 10 About This Instrument Artisan Technology Group Quality Instrumentation
20. 3 10 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Performing a Plate Check Plate Check See Using the Instrument in Chapter 1 for a description of the plate check and its Procedure purpose WARNING ELECTRICAL SHOCK HAZARD Severe electrical shock can result if you defeat the safety interlocks located in the door of the instrument Close the panel door before operating the instrument To check the plates Step Action 1 Open the Special menu and choose Restart to restart the computer We recommend restarting the computer once a day or before each run to reduce memory fragmentation and to quit any applications that might be running in the background If not launched automatically launch the ABI PRISM Data Collection Software data collection software by double clicking the icon Open a new run sheet also referred to as the run window as follows a Open the File menu and select New b Select Sequence Run or GeneScan Run as appropriate c Open the Plate Check Module pop up menu and select a plate check module Click Plate Check on the run sheet The Log and Scan windows are displayed The plates are scanned without electrophoresis Observe the Scan window After approximately 45 seconds four lines are displayed The typical pattern from top to bottom is red black blue and green The blue and
21. A rtisan Artisan Technology Group is your source for quality TecmoogyGroup new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment 7 EQUIPMENT DEMOS HUNDREDS OF InstraV ea REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED D a gaa tia Contact us 888 88 SOURCE sales artisantg com www artisantg com ABI PRism 377 DNA Sequencer For Sequencing and GeneScan Analysis Software Applications User s Manual Applied KS Biosystems Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Copyright 2000 Applied Biosystems For Research Use Only Not for use in diagnostic procedures NOTICE TO PURCHASER This instrument Serial No is Authorized for use in DNA sequencing and fragment analysis This authorization is included in the purchase
22. Guaranteed 888 88 SOURCE www artisantg com ABI PRISM 377 DNA Sequencer System Components Hardware Software Models Available The ABI PRISM 377 DNA Sequencer consists of two main components The ABI PRISM 377 DNA Sequencer the instrument A Macintosh computer A printer is available as optional equipment The components are connected by an asynchronous RS 232C serial data line The ABI PRISM 377 DNA Sequencing System is shipped with the following software ABI PRISM Data Collection Software Data analysis software one or both of the following ABI Prism GeneScan Analysis Software ABI PRISM DNA Sequencing Analysis Software The type of data analysis software shipped with the instrument is determined when the system is ordered Symantec AntiVirus for Macintosh SAM The Norton Utilities The software is installed onto the computer during system installation IMPORTANT Complete and return any registration cards included with the software to ensure you receive notification of future updates Four models of the ABI PRISM 377 DNA Sequencer are available The hardware for each model is identical The data collection software and firmware are different ABI PRISM 377 DNA Sequencer The standard ABI PRISM 377 instrument is designed to accommodate three sizes of gel plates 12 and 36 cm for GeneScan applications and 36 and 48 cm for sequencing applications Combs available are 18 24
23. Plate Check Click the Plate Check button to start a plate check The Plate Check button becomes active when the plate check module is selected on the run sheet The duration of the plate check is specified in the module However the plate check can be paused or cancelled by clicking the Pause or Cancel button PreRun Click the PreRun button to start a prerun The PreRun button becomes active when the prerun module is selected on the run sheet The duration of the prerun is specified in the module A prerun can be paused or cancelled by clicking the Pause or Cancel button Run Click the Run button to start the run The Run button becomes active when the run module and sample sheet are selected on the run sheet Pause Resume The Pause button becomes active when a plate check prerun or run is started Click Pause to temporarily halt instrument operation Pausing a prerun or run halts electrophoresis while maintaining gel temperature When the Pause button is selected it changes to Resume Click Resume to resume instrument operation Cancel The Cancel button becomes active when a plate check prerun or run is started Click Cancel to cancel a plate check prerun or run If cancel is selected during a run the following dialog box is displayed A Do you really want to terminate the run in progress Terminate Stop amp Analyze Continue Clicking Terminate terminates the run without au
24. Plate Check A Plate Check A CHILLER Plate Check B Plate Check B CHILLER Plate Check C Plate Check C CHILLER Plate Check D Plate Check D CHILLER Plate Check F Plate Check F CHILLER Seq PR 36A 1200 Seq PR 36A 1200 CHILLER Seq PR 36B 1200 Seq PR 36B 1200 CHILLER Seq PR 36A 2400 Seq PR 36A 2400 CHILLER Seq PR 36B 2400 Seq PR 36B 2400 CHILLER Seq Run 36A 1200 Seq Run 36A 1200 CHILLER Seq Run 36B 1200 Seq Run 36B 1200 CHILLER Seq Run 36A 2400 Seq Run 36A 2400 CHILLER Seq Run 36B 2400 Seq Run 36B 2400 CHILLER Seq Run 36E 1200 Seq Run 36E 1200 CHILLER Seq Run 36E 2400 Seq Run 36E 2400 CHILLER Seq Run 48A 1200 Seq Run 48A 1200 CHILLER Seq Run 48B 1200 Seq Run 48B 1200 CHILLER Seq Run 48E 1200 Seq Run 48E 1200 CHILLER GS PR 12A 1200 GS PR 12A 1200 CHILLER GS PR 12C 1200 GS PR 12C 1200 CHILLER GS PR 12D 1200 GS PR 12D 1200 CHILLER GS PR 12F 1200 GS PR 12F 1200 CHILLER GS PR 12A 2400 GS PR 12A 2400 CHILLER GS PR 12C 2400 GS PR 12C 2400 CHILLER GS PR 12D 2400 GS PR 12D 2400 CHILLER GS PR 12F 2400 GS PR 12F 2400 CHILLER GS PR 36A 1200 GS PR 36A 1200 CHILLER GS PR 36C 1200 GS PR 36C 1200 CHILLER GS PR 36D 1200 GS PR 36D 1200 CHILLER GS PR 36F 1200 GS PR 36F 1200 CHILLER 1 Use virtual filter set E modules with ABI PRism dRhodam
25. Step Action 1 Add freshly made 10 APS and swirl carefully to mix without introducing air bubbles Note Be as accurate and reproducible as possible when making the 10 APS solution Significant variation in this reagent can produce changes in data quality Add TEMED and swirl carefully to mix without introducing air bubbles Immediately pour the gel Allow the gel to polyermize a minimum of 2 hours before use Note 29 1 polyacrylamide gels take a minimum of 2 hours to polymerize sometimes longer Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com PAGE PLUS Gels Protocol and Run Conditions Overview To obtain the longest read length 5 0 Long Ranger from concentrate or Singel and 4 8 PAGE PLUS gels are recommended for 36 cm runs at 1200 scans hr only These gel formulations have not been shown to consistently increase read lengths on 36 cm runs at 2400 scans hr or 48 cm gel runs Conditions For 48 cm gel runs 5 25 PAGE PLUS and 4 75 Long Ranger Singel gels give longer read lengths than 19 1 polyacrylamide gels For 36 cm gel runs at 2400 scans hr use a 4 5 29 1 polyacrylamide gel Ingredients and Run For 36 cm well to read runs 4 8 PAGE PLUS Gel 6 M Urea Ingredient For 50 mL Run Conditions urea 18 0g Use standard 36 cm 1200 scans hr run 40 gel stock solution 6 0 mL modules 10X TBE
26. for further information regarding external cold water baths Refer to Modules on page 9 42 to see a list of all the modules available including chiller modules External Cooler Off Relay 4 External Cooler On and Off can be used to manually control power to an external water bath Cold Boot Instrument Downloads the firmware image to the instrument Use this command if the computer and instrument are communicating but the instrument is not responding appropriately This symptom indicates the firmware image on the instrument is corrupt See Performing a Cold Boot in Chapter 4 for more information Function range of values Function Range of Values Electrophoresis Volts 0 00 to 5 00 kV Electrophoresis Current 0 00 to 60 0 mA Electrophoresis Power 0 to 300 W Laser Power 0 00 to 40 0 mW in 10ths of mW Pump On 20 to 60 C CCD Gaint 1 2 4 or 8 CCD Output CCD Offset 0 to 4096 CCD Output CCD Pixel Position 1 to 512 pixels These parameters can be controlled manually to make them limiting However you should be thoroughly familiar with the principles of electrophoresis before doing so t This parameter amplifies the signal and should not require adjustment Use this only if instrument memory has been reset and the value has been lost Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www a
27. on page 4 32 Troubleshooting 4 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Observation Possible Causes Recommended Actions Error message The A2D Converter is Not Functioning Converter not functioning No data being transmitted from instrument to computer Turn the instrument power switch off and then on again If the message appears again perform a plate check to see if a signal is displayed If no signal is displayed call service If a signal is displayed and the reoccurrence of this error is not severe you should be able to run samples and collect data If the problems persists call service Flow switch sticks and plugs up Clog in coolant system Flush the coolant system to remove the clog and refill the water reservoir with deionized water and 5 0 antifreeze See Removing Coolant System Clogs on page 4 32 High resistance to flow through heat plates Clog in coolant system Flush the coolant system to remove the clog and refill the water reservoir with deionized water and 5 0 antifreeze See Removing Coolant System Clogs on page 4 32 Quick disconnect fittings leak Buildup behind the seals Flush the coolant system to remove the clog and refill the water reservoir with deionized water and 5 0 antifreeze See Removing Coolant System Clogs on page 4 32 Pump shuts down d
28. www artisantg com Method 1 Part 1 Mounting Glass Plates into the Gel Cassette Using Unmounted Gels can be prepared using mounted or unmounted plates The following procedure is Plates for using mounted plates Refer to Method 2 Pouring the Gel Using Unmounted Plates on page 2 20 if you wish to prepare the gel using unmounted plates Carrying a Cassette with Plates Materials Required Before Mounting the Plates 2 10 Pouring Gels When carrying plates mounted in the gel cassette always hold the cassette by the sides with both hands Do not carry the cassette with plates by holding the top bar only The weight of the plates may cause the bar to break resulting in broken glass plates Do not hold cassette here to carry glass plates x gt gt gt gt o Hold cassette by the sides with both hands to carry glass plates Comb sharks or square tooth Gel cassette Glass plates front and rear one set Kimwipes Spacers two 0 2 mm Water distilled and deionized Before mounting the glass plates into the gel cassette verify that A clean level working area such as a bench top is available for mounting the plates and pouring the gel The plates spacers and comb are clean and dry cleaning procedure on page 2 6 The spacers are the same length
29. Action 1 Wrap the threads of the intake and outlet ports on the external water bath with Teflon tape Attach a metal tubing connector to each port Connect the free end of the intake tube to the outlet fitting on the back of the external water bath Connect the free end of the outlet tube to the intake fitting on the back of the external water bath Subambient Temperature Operation B 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Operating at Subambient Temperatures Performing a Run at Follow these steps to perform runs at subambient temperatures Subambient Temperatures Step Action 1 Follow all instructions in the external water bath user manual 2 Fill the external water bath with cold deionized distilled water and add antifreeze as recommended by the manufacturer WARNING CHEMICAL HAZARD Antifreeze may cause respiratory tract skin and eye irritation Wear chemical resistant gloves and safety glasses when handling and always use in a well ventilated area Check that all tubing is tightly connected Turn on power to the external water bath start the circulating pump and add water as needed to maintain the proper level IMPORTANT If the water level drops below the level recommended by the manufacturer the circulation pump shuts off and the temperature is not maintained Set the exte
30. CCD Pixel Position Value 4 25 Using Calibration File Make and Send 4 27 Temporary Loss of Signal 4 29 Gel Extrusion 4 29 Performing an Alcoholic KOH Wash 4 30 Performing a 3 M HCI Wash 4 31 Removing Gasket Marks from Glass Plates 4 31 Removing Coolant System Clogs 4 32 Optimizing Electrophoresis Conditions for GeneScan Applications 4 34 Troubleshooting 4 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com For More Troubleshooting Information References For more troubleshooting information refer to these manuals as appropriate for your application GeneScan Reference Guide ABI 373 and ABI PRISM 377 DNA Sequencers P N 4303188 Automated DNA Sequencing Chemistry Guide ABI 373 and ABI PRISM 377 DNA Sequencers P N 4305080 4 2 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Gel and Gel Image Troubleshooting Guide Observation Possible Causes Recommended Actions Misshapen wells Note If only a few wells are misshapen you can still load the gel using only those lanes with flat well formed wells Suction when comb removed When removing the comb 1 Lay gel flat 2 Pour 1X TBE over comb 3 Remove comb slowly Wet comb or well former Ensure that comb is clean and dry before inserting into gel Note To ease insertion of comb you can wet
31. Enter the starting point for each file The Start At point should be after the primer peak Define the Points value This is the number of points after the start point to be analyzed Making Matrix Files for GeneScan 6 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To generate the matrix file continued Step Action 5 Click OK A successful matrix opens an untitled Matrix Values window with a 4x4 matrix of numerical values CS 377 D Matrix File SS H Reactions B G Y R 0 0028 0 4001 0 0088 0 0427 0 2193 0 0888 Use the Save As command to name and save the matrix file Choose a name that reflects the chemistry the virtual filter set and the run conditions Check the quality of the matrix file by following the procedure below Checking the Quality of a New Matrix File Checking the Quality of a New Matrix File Procedure Check the quality of the matrix file by reviewing the Values in the Matrix Values window Analyzed data of the matrix run Review the matrix values in the Matrix Values window as follows Step Action 1 View the Matrix Values window O 3477 D Matrix File SS H Reactions G v R 0 0088 0 2193 0 0427 0 0888 The numbers on the diagonal Blue against Blue Green against Green etc must all be 1 00 The numbers off the diagon
32. Guaranteed 888 88 SOURCE www artisantg com Reagent Purity Always use ultra pure reagents obtained from a reliable source and high grade distilled deionized water to prepare solutions and wear clean laboratory gloves Filtration of all solutions is essential to remove any particulate matter that may fluoresce or scatter light Reagent purity and associated problems Reagent Problems That Can Occur When Fresh Reagents are Not Used Acrylamide and bisacrylamide Impurities in these reagents can cause Irreproducible gel porosity Deviant mobility Inhibition of polymerization Poor resolution These problems lead to compromised reproducibility Possible contaminants include Acrylic acid The hydrolysis product of acrylamide Copolymerizes with acrylamide and bisacrylamide Causes DNA to migrate slowly resulting in broad diffuse bands poor resolution Linear polyacrylamide Caused by catalytic contaminants in the dry acrylamide monomer Decreases the effective concentration of the acrylamide causing the DNA to migrate faster Ionic contaminants Mostly metals such as iron or copper Can inhibit or accelerate polymerization IMPORTANT Storage guidelines for these reagent are listed under Storing Reagents and Stock Solutions on page A 23 Proper storage is critical for the production of high quality gels WARNING CHEMICAL HAZARD Acrylamide and bisacryla
33. Linkage Mapping 3000 pmol Set Version 2 Must be ordered through Applied Biosystems Custom Oligonucleotide Synthesis Service specify locus name 403061 True Allele PCR Premix 18 mL enough for 2000 rxns 403062 Control DNA CEPH 1347 02 180 uL enough for 150 rxns Parts and Accessories C 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Applied Biosystems Limited Warranty Applied Biosystems warrants to the Customer that for a period ending on the earlier of one year from completion of installation or fifteen 15 months from the date of shipment to the Customer the Warranty Period the ABI PRISM 377 DNA Sequencer purchased by the Customer the Instrument will be free from defects in material and workmanship and will perform in accordance with the specifications for performance set forth at the time of sale in the Instrument Specification Sheet published and maintained by Applied Biosystems the Specifications During the Warranty Period if the Instrument s hardware fails to perform in accordance with the Specifications Applied Biosystems will repair or replace the Instrument so that it meets the Specifications at Applied Biosystems expense This Warranty does not extend to any Instrument or part which has been a the subject of
34. Ln PreRun Module Seq PR 364 2400 w Run Module Seq Run 36A 2400 w EJ Autoanalyze with Other CT Ex Auto Print _ Concer Cox 3 To change the default setting for the WTR distance Lanes PreRun Module and Run Modules open the pop up menu for each parameter and select the desired default 4 To specify that data be automatically analyzed by the sequencing analysis software a Select the box labeled Autoanalyze with b Open the autoanalyze pop up menu select Other c Locate and select double click or OPEN the appropriate analysis application ABI PRISM DNA Sequencing Analysis Software 5 To specify that data be printed automatically at the end of each run select the check box labeled Auto Print Setting Preferences 5 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To change sequencing run file preferences continued Step Action 6 If Then you are finished click OK to save your changes or Cancel to quit without making any changes you wish to change more preferences open the Page pop up menu and select another Preference GeneScan Sample Sheet Preferences Setting GeneScan For GeneScan sample sheets one parameter can be set as a preference the size Sample Sheet standard dye color To change GeneScan sample sheet pref
35. Run temperature is typically reached after 15 25 minutes even though the default duration of the prerun module may be longer Indicates the amount of time left for the specified module to run Status Instrument State mE Running Laser Power mE Running Electrophoresis Power mer On Door mE Closed v r Time Remaining 00 58 29 f Total 01 00 00 Total duration of the module V Electrophoresis Electrophoresis Electrophoresis Gel Laser Voltage kY Current m Power W Temperature C Power mW Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Once the gel reaches run temperature the prerun is typically paused not cancelled and the samples are loaded When paused the pump and heater remain on and the gel temperature designated by the module is maintained Once the samples are loaded the prerun is cancelled and the run is started Instructions for performing a prerun are listed in Chapter 3 Instrument Operation The status window and the run sheet are described in more detail in Chapter 3 Instrument Operation and in Chapter 9 Data Collection Software The Run The run is typically performed after the plate check and prerun During the run samples are electrophoresed and data is collected To perform a run the run sheet must be completed i e the run module sample sheet numb
36. Using Calibration File Make and Send on page 4 27 for more information Troubleshooting 4 25 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Location of the CCD The correct CCD pixel position value is printed on a white label affixed to the CCD Pixel Position Value camera The label is visible from the front of the instrument through the opening below the rear heat transfer plate Rear heat transfer plate CCD camera with pixel position label visible here Locating the CCD To locate the CCD pixel position value Pixel Position Value Step Action 1 Have a flashlight available 2 Open the front door of the instrument 3 Shine the flashlight through the opening below the rear heat transfer plate and locate the white label on the CCD camera Record the value from the white label If you cannot find the label call Applied Biosystems technical support telephone numbers listed in Chapter 1 Checking and To enter the CCD pixel position value Action Turn power on to the instrument Select Manual Control from the Window menu in the data collection software Open the Fxn Name pop up menu and select the CCD Pixel Position function The current pixel position value is displayed If it is the same as the value on the white label do not complete the remaining steps If the value is different con
37. Working in a fume hood combine the appropriate amounts acrylamide and bisacrylamide in a glass beaker WARNING CHEMICAL HAZARD Acrylamide and bisacrylamide are poisons neurotoxins irritants carcinogens and possible teratogens Acrylamide and bisacrylamide sublime the solids release toxic vapor and are harmful if swallowed inhaled or absorbed through the skin Effects are cumulative When handling always wear protective equipment lab coat safety glasses and chemical resistant gloves and use in a well ventilated area On a routine basis thoroughly clean surfaces subject to contamination Dissolve the crystalline acrylamide and bisacrylamide in enough distilled deionized water to bring the total volume to 135 mL Add 15 g of mixed bed ion exchange resin Stir at room temperature until all crystals dissolve Continue stirring for 5 10 minutes Filter the mixture through 0 2 ym cellulose nitrate filter Transfer the filtrate to a graduated cylinder and bring the total volume to 150 mL with distilled deionized water Store at 2 6 C Note 40 polyacrylamide stock lasts for 1 month at 2 6 C Gel Recipes A 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com For All Applications To prepare 40 mL of an N polyacrylamide 6 M urea gel Except SSCP Step Action 1 Instructions for preparing glass plates and two ge
38. analyzed data is printed automatically See also About Automatic Data Analysis on page 9 38 Can be set as Preferences See Setting Run Sheet Preferences on page 9 31 9 30 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Preparing a Run Sheet Setting Run If the same type of run is performed repeatedly on the same instrument you can Sheet Preferences reduce the time spent setting up run sheets by setting sequencing or GeneScan run sheet default preferences Setting run sheet preferences means setting the default value of certain fields on the run sheet template to the values used most often The following fields can be set as preferences Operator Auto gt gt gt gt o o o o Lanes Well to read distance Prerun and run modules Software program to be used for automatic data analysis print Analysis parameters GeneScan run sheets only Gel s matrix file GeneScan run sheets only Size standard GeneScan run sheets only Once these preferences are set the preferred values appear automatically on each new run sheet Preferences can be changed as often as necessary either by setting new preference values or by manually selecting new files values on the run sheet Instructions for setting run sheet preferences are located in Chapter 5 Setting Preferences Opening a New To open a new run shee
39. the comb with acrylamide or the appropriate gel polymer Air bubbles trapped when comb inserted Ensure that no air is trapped by comb Swirls in gel Schlieren pattern Excessive TEMED or APS Prepare new solutions following protocol Temperature too high Polymerize at 20 23 C Gel polymerized too quickly Do not heat gel solution to dissolve urea Insufficient reagent mixing Mix reagents gently but thoroughly Polymerization too slow gels should polymerize within 15 20 minutes Excessive dissolved oxygen Keep vacuum filter strength and time constant Stir and pour solutions gently Filter pour gels at 20 23 C Not enough TEMED or APS Degraded TEMED or APS Prepare new solutions following the protocol using fresh high quality reagents Store APS at room temperature in a desiccator Store TEMED in tightly sealed container at room temperature Temperature too low during casting Polymerize at 20 23 C Did not use deionized water Use only distilled or deionized water for making all solutions Gel solution leaks out when injecting gel Bottom of plates not flush Gel pouring fixtures not mounted correctly Plates not positioned properly in the cassette Review the gel pouring procedures in Chapter 2 Pouring Gels Gel extrudes from between plates into upper buffer chamber during electrophoresis Large migrating region showing
40. to create enough space First back up important files onto floppy disks or another storage device then delete files off the hard disk and click Run again Verify electrophoresis has resumed by viewing the Status window Verify scanning has resumed by checking the following in the Scan window All four scan lines should be above zero Current scan number should increment If either electrophoresis or scanning does not resume cancel the run and restart it If this is a 96 lane instrument load your samples now as follows a Click Pause to pause the run b Open the front panel of the instrument c Carefully flush all of the wells with 1X TBE buffer to remove urea WARNING Tris borate EDTA TBE buffer can be harmful if inhaled ingested or absorbed through the skin It is irritating to the eyes skin and mucous membranes Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Load samples in the odd numbered lanes Click Resume and allow the samples to electrophorese for one minute Click Pause ompa Carefully flush all of the wells with 1X TBE buffer to remove any residual formamide from the previously loaded wells h Load the remaining samples i Close the front panel of the instrument j Click Resume to continue the run To cancel a run in progress and analyze the data collected up to that point click Cancel and then click Stop amp
41. www appliedbiosystems com AR Bes ys D Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 08 2001 Part Number 4307164B an Applera business Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Subambient Temperature Operation Appendix Contents In this Appendix The information in this appendix describes how to modify your ABI PRISM 377 DNA Sequencer to run with an external gel cooling system water bath for run temperatures below the standard temperature The standard run temperature is ambient plus 10 C up to 60 C The following topics are discussed in this appendix Topic See page Purpose of an External Cooling System B 1 Hardware and Software Requirements B 2 How the External Water Bath Functions B 3 Installing the External Water Bath B 5 Operating at Subambient Temperatures B 6 Avoiding Condensation Inside the Instrument B 7 For More Refer to the GeneScan Reference Guide ABI 373 and ABI PRISM 377 DNA Information Sequencers P N 4303188 for more information on Single stranded Conformation Polymorphism SSCP and operating the instrument at subambient temperatures Purpose of an External Cooling System Performing SSCP The ABI PRISM 377 DNA
42. 0 0 3 3 Preparing the Formamide Blue Dextran Loading Solution 0 3 3 Cleaning the Gel Plates Before Loading the Gel 1 0 0 cee eee ee eee 3 4 Loading the Gel Into the Cassette 2 ccc eee ences 3 6 Installing the Gel Cassette and Lower Buffer Chamber 0 0 00 0000 ee 3 8 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Pe rforming a Plate Check ccs carsce co ee ee ee See he ba eee 3 11 Skipping MANS cs csi yes evdeis seca nET cad as aess Pesos Me anbede kere nes 3 14 Installing the Upper Buffer Chamber 20 0 0 cece eee eee ee 3 15 Filling the Buffer Chambers 0 0 0 cece cece eee eee 3 16 Installing the Front Heat Transfer Plate 2 0 0 eee eee 3 18 Setting Up the Software fora Run 2 2 ce eect eens 3 19 Preparing a Sequencing Sample Sheet 0 0 2c eee cece 3 19 Preparing a GeneScan Sample Sheet 0 0 0 cee eee 3 24 Preparing a Ruin Sheet iss ses eae eth eed ea ee dee ee a 3 28 Performing a PreRun and Loading the Samples 0 0 0 eee eee eee 3 35 Starting and Monitoring the Run 0 2 cc eee eens 3 40 Viewing the Log Pile oc tiiniiee neies iiie E ian oe eee antlers 3 44 Cleaning Up After the RUN cecce irsi ner Seba eee eee ed eee eee ee Pe ede 3 45 Analyzing the Data cn viGievede vibes ohege td AGG eA eased aes eden geeh helped 3 46 Archiving and Printi
43. 2 Click the box that corresponds to the folder location you wish to set Preferences Page Folder Locations vj 7 Sample Sheet Folder Macintosh HD ABI Prism 377 Sample Sheets CA Module Folder Macintosh HD ABI Prism 377 Modules C Folder Containi ng Run Folders Macintosh HD ABI Prism 377 Runs CI Firmware File Folder Macintosh HD ABI Prism 377 Firmware Image fa Settings Folder Macintosh HD System Folder ABI Folder A GeneScan Analysis Parameters Macintosh HD GeneScan G3 Parameters Folder 4 CA GeneScan Size Standard Folder Macintosh HD GeneScan 0 65 Standards Folder Co Click in these boxes to select and change a folder location Displayed for GeneScan users only 5 6 Setting Preferences Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To set the Folder Location preferences continued Step Action 3 Using the pop up menu in the dialog box that is displayed locate and select the appropriate folder by clicking the folder once only IMPORTANT To automatically analyze data with ABI PRISM DNA Sequencing Analysis Software on the same computer used for data collection the Settings Folder must be designated as the ABI Folder shown below 1 The text above the menu indicates the folder location pre
44. 251 24 243 France Paris 33 0 1 69 59 85 85 33 0 1 69 59 85 00 Germany Weiterstadt 49 0 6150 101 0 49 0 6150 101 101 Hungary Budapest 36 0 1 270 8398 36 0 1 270 8288 Italy Milano 39 0 39 83891 39 0 39 838 9492 Norway Oslo 47 23 12 06 05 47 23 12 05 75 Poland Lithuania Latvia and Estonia 48 22 866 40 10 48 22 866 40 20 Warszawa Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Russia Moskva 7 095 935 8888 7 095 564 8787 South East Europe Zagreb Croatia 385 1 34 91 927 385 1 34 91 840 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 1206 Sweden Stockholm 46 0 8 619 4400 46 0 8 619 4401 Switzerland Rotkreuz 41 0 41 799 7777 41 0 41 790 0676 The Netherlands Nieuwerkerk a d IJssel 31 0 180 331400 31 0 180 331409 United Kingdom Warrington Cheshire 44 0 1925 825650 44 0 1925 282502 All other countries not listed Warrington UK 44 0 1925 282481 44 0 1925 282509 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Telephone Fax Region Dial Dial Japan Japan Hacchobori Chuo Ku Tokyo 81 3 5566 6230 81 3 5566 6507 Latin America Del A Obregon Mexico 305 670 4350 305 670 4349 To Reach Technical We strongly encourage you to visit our Web site
45. 37 Selecting Auto Analyze allows you to select analysis parameters a size standard and the auto print option If matrix standard samples are being run deselect Auto Analyze for all matrix standard samples If not already selected or if you wish to change the preference settings set the remaining fields as follows a Open the Analysis Parameters pop up menu and select an analysis parameters file Analysis parameters must be set up in the analysis software program prior to completing the run sheet Refer to ABI PRISM GeneScan Analysis Software User s Manual for instructions b Open the Size Standard pop up menu and select the appropriate size standard file c Select the Auto Print boxes for analyzed data to be printed automatically Note If the pop up menus list lt none gt only software cannot find the folders that contain the analysis parameters and size standard files To correct this see Commands Used to Perform a Plate Check PreRun and Run on page 9 39 9 36 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Auto analyze deselected This Auto analyze selected A set of automatically deactivates the analysis parameters and a size analysis parameters size standard must be selected standard and auto print fields hun Sheet Example gt Plate Check Module Run Modu
46. 5 0 mL Increase run time to 9 hr deionized water to 50 0 mL 10 APS 300 uL TEMED 30 0 pL For 48 cm well to read runs 5 25 PAGE PLUS Gel 6 M Urea Ingredient For 50 mL Run Conditions urea 18 0g Use 48 cm run module 40 gel stock solution 6 6 mL Increase run time to 12 hr 10X TBE 5 0 mL deionized water to 50 0 mL 10 APS 250 uL TEMED 25 0 uL Gel Recipes A 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Protocol for To prepare 4 8 and 5 25 PAGE PLUS gels PAGE PLUS Gels A 14 Gel Recipes Step Action 1 Instructions for preparing glass plates and two gel pouring methods are located in Chapter 2 Pouring Gels Glass plates and all other gel pouring equipment must be ready for use prior to adding the polymerizing reagents to the gel solution Prepare all stock solutions per the appropriate list of ingredients on page A 13 Weigh out the urea and carefully transfer it to a stoppered graduated cylinder Using a pipette add the appropriate amount of gel stock solution and 10X TBE buffer to the cylinder Adjust the volume to 49 5 mL by slowly adding deionized water and tapping the cylinder to release air bubbles trapped by the urea Stopper the cylinder and invert to dissolve the urea Allow the solution to warm to room temperature Add deionized water to bring the s
47. 57 warranty D 1 ABI Prism 377 DNA Sequencer with 96 Lane Upgrade description of instrument 1 12 ABI Prism 377 DNA Sequencer with XL Upgrade description of instrument 1 11 to 1 12 ABI Prism 377 Site Preparation and Safety Guide description of guide 1 4 ABI Prism 377 18 DNA Sequencer description of instrument 1 11 ABI Prism DNA Sequencing Software automatic data analysis 9 38 description of software 1 19 instrument files adding replacing matrix 7 20 to 7 21 color guide for data display 7 4 to 7 5 for Virtual Filter Set A 7 6 to 1 11 to 7 9 for Virtual Filter SetE 7 10to 7 15 from a sample file 7 18 to 7 19 overview 7 2 storing and backing up 7 19 summary of chemistries 7 4 terminology 7 1 verifying instrument files 7 16 to 7 17 when to make new file 7 3 run sheets about 9 28 to 9 29 preparing 9 31 to 9 37 setting preferences 3 28 5 11 9 31 Index 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com sample sheets about 9 14 to 9 15 entering information 9 26 importing exporting information 9 27 preparing 9 16 to 9 19 setting preferences 3 19 5 10 9 16 ABI Prism GeneScan Analysis Software about automatic data analysis 9 38 description of software 1 18 fragment kits and reagents available C 8 to C 11 matrix files creating a matrix 6 6 to 6 8 evaluating the quality of 6 3 to 6 5 purpose of 6 2 terminology 6 1 verifying new matrices 6 8 to 6 9 wh
48. 8 Rinse the plates thoroughly with distilled deionized water Allow plates to dry Note Avoid other cleaning procedures or solutions that may reintroduce contaminants to the plates 4 30 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Performing a 3 M HCI Wash Procedure To perform a 3 M HCI wash Step Action 1 Place some uncolored absorbent towels or other covering in the hood to catch spills Pour 10 mL of concentrated HCI 12 N 37 carefully into 30 mL of water and mix thoroughly WARNING Hydrochloric acid HCl is a very corrosive liquid Always work in a fume hood to avoid inhalation Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Place the plates on the towels with the inside surfaces facing up Note The plates should be nearly level so that the cleaning solution does not run off onto the bench Only the inside gel side surface of the plates need be cleaned though the outside surfaces can be cleaned similarly Pour approximately 15 mL of the cleaning solution in the center of each plate to be cleaned Spread the solution over the surface of plate Allow the solution to remain on the plates for 5 minutes Note Longer times will not harm the plates but are unnecessary Rinse the plates thoroughly with distilled deionized water Allow
49. ABI PRISM 377 DNA Sequencer ABI PRISM BioLIMS software was designed by Applied Biosystems in collaboration with Molecular Informatics a key player in the development of the Genome Sequence Database the world s most widely used relational database for genetic sequence information It is the first commercially available system for automated genetic information management and an excellent foundation on which to build your current and future bioinformatics capabilities Using BioLIMS software you can automate DNA sequencing from data acquisition to assembled contig Data flows automatically from your ABI PRISM 377 DNA Sequencer into the ABI PRISM BioLIMS relational database and on to downstream analysis packages To simplify project tracking and automation all sequences are stored by project name Using UNIX and AppleScript to control the process sequence information flows seamlessly from your instruments to the database and into the analysis packages ABI PRISM GeneScan Analysis Software BioLIMS version 2 0 and up only ABI PRISM Sequencing Analysis Software Factura and AutoAssembler programs all read and write to the database Just specify the sequences you want to analyze and the applications you want to use The ABI PRISM BioLIMS software system simplifies data retrieval analysis and reporting It maintains data integrity by eliminating multiple copies of the same data and it helps improve reporting efficiency by allowing y
50. Adding Non Applied Biosystems Software on page 8 28 for more information Note Store the System floppy disks or CDs in a safe place We also recommend making a copy of each disk A utility such as Disk Copy can be used For more information on the System file and folder refer to the Macintosh system reference manual System Maintenance 8 29 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Data Collection Software Chapter Contents In this Chapter The following topics are discussed in this chapter Topic See page System Software Overview 9 2 Data Collection Software Files and Folders 9 3 Setting Folder Location Preferences 9 5 Data Flow Between the Computer and Instrument 9 7 Launching the Data Collection Software Program 9 8 Menu Commands 9 9 About Sample Sheets for Sequencing Applications 9 14 Preparing a Sequencing Sample Sheet 9 16 About Sample Sheets for GeneScan Applications 9 20 Preparing a GeneScan Sample Sheet 9 22 How to Enter Information on Sample Sheets 9 26 Importing and Exporting Sample Sheet Information 9 27 About Run Sheets 9 28 Preparing a Run Sheet 9 31 About Automatic Data Analysis 9 38 Commands Used to Perform a Plate Check PreRun and Run 9 39 Modules 9 42 DyeSe
51. Box Matrix Taq Terminator Matrix Matrix C dR110 dROX dR6G A dR6G dR6G dTAMRA G dTAMRA dR110 dROX T dROX dTAMRA dR110 Perform the following procedures to make an instrument file for Filter Set E dRhodamine based chemistries Making the Dye Primer Matrix on page 7 11 Making the Taq Terminator Matrix on page 7 14 Making the T7 Terminator Matrix on page 7 15 When finished check the instrument file by following the procedure Making an Instrument File for Virtual Filter Set E from Matrix Standards on page 7 10 7 10 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Making the Dye Primer Matrix To make the Dye Primer Matrix Step Action 1 Launch the Data Utility software located in the Utilities folder within the Sequencing Analysis folder The icon looks like this From the Utilities menu choose Make Matrix The Make Matrix dialog box appears as shown below Verify that the Dye Primer Matrix button at the lower left is selected Make Matrix Start at Start at Start at Start at Points Dye Primer Matrix Tag Terminator Matrix T Terminator Matrix Click on the box for each nucleotide base and select the sample file that corresponds to the correct matrix standard as shown in the table below Dye Primer Box Matrix d
52. Click OK 9 56 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Viewing Other The Sample Sheet Run Related To display the sample sheet associated with the current run choose Sample Sheet Information from the Window menu This option is available only if a sample sheet is selected in a Run window that is open For more information see About Sample Sheets for Sequencing Applications on page 9 14 About Sample Sheets for GeneScan Applications on page 9 20 Sequencing or GeneScan Analysis Software user s manual as appropriate The Run Sheet To display the Run sheet for the current run choose Run from the Window menu For more information see About Run Sheets on page 9 28 Opening and Saving Files Opening Files Most files are opened using the Open command in the File menu Other files such as Log and Gel files are opened by clicking the file icon from the data collection software program Saving Files Files are saved by choosing one of the Save commands from the File menu Sample and run sheets are automatically named and saved based on the default settings entered for the Default File Names and Folder Locations Preferences Refer to Chapter 5 Setting Preferences for more information When modifying existing sample sheets run sheets or Module Settings dialog boxes we recommend saving the changes under
53. Folder Macintosh HD Contains all the sample sheets When ABI Prism 377 setting up a run sheet the selections Sample Sheets displayed on the pop up menu for sample sheets are the files stored in this folder Module Folder Macintosh HD Contains the Plate Check PreRun and Run ABI Prism 377 modules When setting up a run sheet the Modules selections displayed on the Plate Check PreRun and Run Module pop up menus are the files stored in this folder Chiller Modules Macintosh HD Contains chiller Plate Check PreRun and Folder ABI Prism 377 Run modules When setting up a run sheet Chiller Modules the selections displayed on the Plate Check PreRun and Run Module pop up menus are the files stored in this folder Folder Containing Macintosh HD Contains the Run folders A new Run folder Run Folders ABI Prism 377 is created for each run At the end of data Runs analysis each new Run folder contains a gel file log file run sheet and sample files Firmware File Folder Macintosh HD Contains the firmware image file ABI Prism 377 Firmware Image IMPORTANT Locations and folder names listed are those at the time of system installation We strongly recommend not changing folder names and locations Displayed in Folder Locations dialog box for GeneScan users only Setting Preferences 5 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com
54. Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Injecting the WARNING CHEMICAL HAZARD Long Ranger gel solution contains acrylamide Gel Solution Acrylamide is a neurotoxin Avoid skin contact with Long Ranger gel solution because acrylamide can be absorbed through the skin Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves WARNING CHEMICAL HAZARD Acrylamide and bisacrylamide are poisons neurotoxins irritants carcinogens and possible teratogens Acrylamide and bisacrylamide sublime the solids release toxic vapor and are harmful if swallowed inhaled or absorbed through the skin Effects are cumulative When handling always wear protective equipment lab coat safety glasses and chemical resistant gloves and use in a well ventilated area On a routine basis thoroughly clean surfaces subject to contamination To inject the gel solution Step Action 1 Draw at least 35 mL of gel solution into a 60 cc syringe filling it slowly to avoid introducing air bubbles IMPORTANT Draw enough solution into the syringe the first time to inject the entire gel without having to stop and draw more solution 2 Check the tip of the syringe The gel solution should be at the tip and no air bubbles should be present Adjust the solution and remove air bubbles if necessary 3 Starting at one end of the lar
55. Guaranteed 888 88 SOURCE www artisantg com Product or Product Area Telephone Dial Fax Dial ABI Prism 3100 Genetic Analyzer 1 800 831 6844 then press 26 1 650 638 5981 Biolnformatics includes BioLIMS BioMerge and SQL GT applications 1 800 831 6844 then press 25 1 505 982 7690 Peptide Synthesis 433 and 43X Systems 1 800 831 6844 then press 31 1 650 638 5981 Protein Sequencing Procise Protein Sequencing Systems 1 800 831 6844 then press 32 1 650 638 5981 PCR and Sequence Detection 1 800 762 4001 then press 1 for PCR 2 for the 7700 or 5700 6 for the 6700 or dial 1 800 831 6844 then press 5 1 240 453 4613 Voyager MALDI TOF Biospectrometry and Mariner ESI TOF Mass Spectrometry Workstations 1 800 899 5858 then press 13 1 508 383 7855 Biochromatography BioCAD Workstations and Poros Perfusion Chromatography Products 1 800 899 5858 then press 14 1 508 383 7855 Expedite Nucleic acid Synthesis Systems 1 800 899 5858 then press 15 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 then press 15 1 508 383 7855 PNA Custom and Synthesis 1 800 899 5858 then press 15 1 508 383 7855 FMAT 8100 HTS System and Cytofluor 4000 Fluorescence Plate Reader 1 800 899 5858 then press 16 1 508 383 7855 Chemilumi
56. Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Pouring Gels Chapter Contents In this Chapter The following topics are discussed in this chapter Topics See page Selecting a Gel Formulation 2 2 Importance of Using High Quality Gels 2 3 About the Gel Pouring Methods in This Chapter 2 4 Before Using New Glass Plates 2 5 Cleaning Glass Plates Spacers and Combs 2 6 Method 1 Part 1 Mounting Glass Plates into the Gel Cassette 2 10 Method 1 Part 2 Attaching the Gel Injection Device 2 14 Method 1 Part 3 Pouring the Gel Using Mounted Plates 2 18 Method 2 Pouring the Gel Using Unmounted Plates 2 20 Related Information in Appendix A Gel Recipes Topics in The following related topics and information are located in Appendix A Gel Recipes Appendix A Topics Gel recipes and recommendations for ABI PRISM DNA Sequencing and GeneScan analysis software applications How to store reagents and stock solutions Factors that affect sequencing read lengths Factors that affect gel quality including Purity of reagents Rate of polymerization Air bubbles Age of the gel Pouring Gels 2 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Selecting a Gel Formulati
57. It is believed to be caused by a buildup of charge on the surface of the glass plate such that the gel is not bound to the plate after pouring As the voltage is applied the gel migrates toward the upper electrode The gel image can show a variety of anomalous effects including catastrophic loss of resolution lane splitting extreme band tilt and band distortion Almost all known cases of gel extrusion have been resolved by alcoholic KOH washing or acid washing See Performing an Alcoholic KOH Wash on page 4 30 and Performing a 3 M HCI Wash on page 4 31 Troubleshooting 4 29 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Performing an Alcoholic KOH Wash Procedure The following procedures are not meant to be used for regular gel plate maintenance but for decontamination For regular plate cleaning we recommend using a dishwasher with a hot deionized water rinse WARNING Preparation of all solutions should be carried out in a hood using safety glasses gloves and other appropriate protective clothing To perform an alcoholic KOH wash Step Action 1 Add 30 35 g of potassium hydroxide KOH or sodium hydroxide NaOH pellets to a plastic bottle WARNING Potassium hydroxide is hygroscopic and caustic It can cause severe burns and blindness if it comes in contact with the skin or eyes Always work in a fume hood Obtain a copy of the MSDS from
58. Material Data Safety Sheets for the chemicals commonly used with the instrument We strongly recommend this guide be kept readily available for reference at all times Safety warnings appear throughout this manual at relevant locations These warnings address chemical high voltage and laser safety They appear in the format shown below WARNING ELECTRICAL SHOCK HAZARD The ABI Prism 377 contains a high voltage power supply Although the instrument has been designed with safety features in the door to disconnect the power supply when the door is open please follow procedures as prescribed As with any electrophoresis apparatus be careful during instrument operation and when handling electrodes and liquids WARNING LASER HAZARD Exposure to direct or reflected laser light at 40 mW for 0 1 seconds can burn the retina and leave permanent blind spots Never look directly into the laser beam or allow a reflection of the beam to enter your eyes WARNING Ergonomic Hazard Performing loading activities may increase risk of developing the following cumulative trauma disorders repetitive motion or repetitive strain injuries which include but are not limited to tendinitis tenosynovitis epicondylitis strains and or sprains To reduce the risk of experiencing these types of disorders the following recommendations have been developed to decrease awkward posture repetitive motion excessive force static muscle loading and soft tissue c
59. Refer to the SAM user s manual for more detailed information System Maintenance 8 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Norton Utilities Product Overview Norton Utilities is a collection of software utilities designed for data protection and recovery Use the following utilities to repair or recover disks folders or files Norton Disk Doctor repairs hard disks Volume Recover restores accidentally initialized but not formatted hard disks or badly damaged disks using FileSaver information UnErase Recovers files that have been thrown away or are not visible due to disk damage Norton Disk Editor Edits data directly on the hard disk Use the following utilities to prevent disk problems from becoming serious and to minimize the effects of unavoidable damage FileSaver Checks disks for problems while the Macintosh is idle and alerts the user to problems as they develop Tells users when to perform backups or optimization Saves valuable information about your disks and files that other Norton Utilities use Startup Disk Builder creates three types of custom startup disks used to start up the Macintosh and run Norton Disk Doctor Norton Disk Editor and Speed Disk Norton Fastback Makes and restores backup copies of your files Wipe Info Overwrites file contents so that they can never be recovered DiskLigh
60. SOURCE www artisantg com To change Dye Indicator preferences continued Step Action 4 If Then Other is not selected proceed to step 6 Other is selected a use the Hue Angle and Saturation scroll bars fields or the color wheel as described in the illustration to change the hue and saturation b Use the Lightness scroll bar field or the lightness bar as described in the illustration to make the color lighter or darker As changes are made the new color is displayed in the New box c When finished choose one of the following Click OK to save the change and return to the Dye Indicators preference window Click Cancel to cancel the change and return to the Dye Indicators preference window Click More Choices and proceed to step 5 To use the wheel move cursor onto wheel Cursor turns to crosshair Click and drag the Color before changes are made Original Color as you make New changes Use the wheel or these boxes to change hue and Saturation i saturation Hue Angle Use the bar or this box to change the lightness Lightness Click and drag this bar to make changes 5 18 Setting Preferences Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To change Dye Indicator preferences continued Step Action 5 If Then More Choices is not
61. Sequencer controls gel temperature from 10 C above ambient temperature to a maximum of 60 C To perform PCR Single stranded Conformation Polymorphism SSCP protocols at lower temperatures below 10 C above ambient a water bath must be attached to the instrument For more information on performing SSCP refer to PCR SSCP Analysis A Guide to Fluorescent PCR Single stranded Conformation Polymorphism Analysis on the ABI PRISM 377 DNA Sequencer P N 904413 Subambient Temperature Operation B 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Hardware and Software Requirements Overview The following hardware and software is required to operate the instrument at subambient temperatures Anexternal water bath connected to the instrument 30 k ohm thermistors installed on the instrument ABI PRISM 377 Data Collection Software version 2 1 or higher Hardware Water Bath Requirements The water bath polyurethane tubing and tubing connectors required for instrument modification must meet the following minimum requirements Hardware Minimum Requirements Water Bath Cooling capacity 250 watts at 20 C Heating capacity 500 watts up to 60 C Pump flow rate 1 gal min or 4 L min at pressure head of 3 m Automatic Shutdown At high temperature and low liquid level Polyurethane Tubing 6 ft 1 4 in i d 3 8 inch o d Tubing Connectors 1 4 in female
62. Set virtual filter set E Any Primer works with any primer DT4 Ac B Set AnyPrimer DT dye terminator 4 Ac 4 acrylamide gel B Set virtual filter set B Any Primer works with any primer Choosing a DyeSet The dye set primer files listed below are recommended for the following sequencing Primer File chemistries Dye Set Primer Files and DNA Sequencing Chemistries Dye Primer BigDye Primer dRhodamine Dye Terminator Terminator BigDye Terminator DP4 Acv2 M1 DP5 LR BD M13 DT4 Ac A DT dR Set DT BD Set 3Rev FWD amp REV DP4 Ac 21M 13 DP4 Ac KS DP4 Ac SK DP4 Ac SP6 DP4 Ac T3 DP4 Ac T7 Set AnyPrimer Any Primer Any Primer If the wrong dye set primer file is specified on the sequencing sample sheet and is used for automatic data analysis the data can be reanalyzed using the correct file Refer to the Automated DNA Sequencing Chemistry Guide for more information 9 50 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Virtual Filter Sets What Are They Virtual filter sets are used to detect light intensity in four non overlapping light collecting regions on a CCD camera located inside the instrument Each region corresponds to a wavelength range that contains or is close to the emission maximum of an ABI Prism dye Data collection software color codes the intensity displays from
63. System folder Do not delete Created in the these files They are deleted automatically when no longer needed System Folder Launching the Data Collection Software Program How to Launch To launch the data collection program Step Action 1 From the Finder open the Special menu and select Restart We recommend restarting the computer once a day or before each run to reduce memory fragmentation and to quit any applications running in the background Double click the data collection software program icon or the alias AGI Prism 377 Colection If Then the software launches properly proceed with preparing the instrument and software for a run a dialog box asking for folder locations appears you must set the Folder Locations Preference Instructions are located on page 9 5 and in Chapter 5 Setting Preferences 9 8 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Menu Commands Overview Active versus Inactive Menu Commands Apple Menu Data collection software has its own menu bar shown below located at the top of the computer screen after the software is launched Pull down menus and the point and click technique are used to execute commands As you become familiar with this software you can also use shortcut keyboard commands when available Shortcut keyboard com
64. Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com www appliedbiosystems com AR Bes ys D Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 08 2001 Part Number 4307164B an Applera business Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com A rtisan Artisan Technology Group is your source for quality TecmoogyGroup new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sel
65. Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Observation Signal too high streaks appearing or peaks heights greater than 4000 RFU Possible Causes Too much sample added to some or all lanes Unincorporated F dNTPs Recommended Actions Load less sample Reamplify using less F JdNTPs Purify the PCR product Unincorporated dye terminators Follow the protocols for excess dye terminator removal carefully See the user bulletin Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions P N 4304655 Obtain from our website at www appliedbiosystems com techsupp ort High noisy baseline Matrix made incorrectly resulting in too much correction also indicated by troughs under peaks Remake matrix Be sure to Remove primer peak or aberrant off scale peaks from scan range Pick start and stop points on flat parts of the baseline when viewing raw data Make matrix using same gel formulation buffer and run conditions as samples Contamination of buffer with fluorescent species for example from laboratory pen used to label lanes gel plates or comb Do not label gel plates or comb Lower buffer chamber not clean i e contaminated with fluorescent species Clean and replenish buffer in lower buffer chamber Signal gets progressively weaker for successive gels run over time 4 12 T
66. The Preferences dialog box in the specific analysis program allows you to choose the file format Other files provide information for a run that you might have customized and should therefore save These include the sample sheets analysis settings or the matrix instrument files you create System Maintenance 8 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Sample Sheet Files Sample sheets can be a good reference after data collection Individually sample sheet files are small enough to archive on floppy disks Custom Analysis A backup copy of all custom analysis settings and matrix instrument files should be Settings and kept on a separate storage medium Each of these types of files alone is small enough Matrix Files to store on a floppy disk 8 24 System Maintenance Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Reinstalling ABI PRISM 377 Software System Software The following software is supplied with the ABI PRism 377 and is installed onto the computer by the Applied Biosystems Field Service Specialist during system installation ABI PRISM Data Collection Software version 2 1 or higher Data analysis software one or both of the following ABI PRISM GeneScan Analysis Software version 2 1 or higher ABI PRISM DNA Sequencing Analysis Software version 3 0 or higher Symantec AntiVirus for Ma
67. Undo Typing Undo Paste Can t Undo Cut Copy Standard Macintosh commands Refer to the Macintosh manual for more information Paste Clear Show Clipboard Select All Selects all the cells of a sample sheet Fill Down Inserts text into all the cells selected in a column on a sample sheet Set Scale Sets the scale of the panels in the Electrophoresis History window Show Clipboard The information selected when using the Copy command is saved to the clipboard Use this command to see what you have copied to the clipboard prior to using the Paste command Data Collection Software 9 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Instrument Menu mer Windou Start Plate Check Start PreRun Start Run Pause Resume The Instrument menu contains commands for starting pausing and stopping instrument operation Cancel Run Instrument menu commands For more Command Description detail see Start Plate Check Starts a plate check using the settings of the plate check module specified on the run sheet The gel and plates are scanned without electrophoresis before loading samples to ensure the plates are clean and the gel is not contaminated Start PreRun Starts a prerun using the settings of the prerun module specified on the run sheet Start Run Starts a run using the settings of the run module Chapters speci
68. User s Manual Software 4303242 ABI Prism GeneScan Analysis Software User s Manual version 3 0 902842 GeneScan 672 Software User s Manual 4303188 GeneScan Reference Guide ABI 373 and ABI PRISM 377 DNA Sequencers 4305080 Automated DNA Sequencing Chemistry Guide ABI 373 and ABI PRISM 377 DNA Sequencers 904532 ABI PrisM DNA Sequencing Software User s Manual version 3 0 4304075 ABI PRISM DNA Sequencing Software User s Manual version 3 2 Parts and Accessories Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com C 7 ABI PRISM DNA Fragment Analysis Kits and Reagents GeneScan Size Standards GeneScan 350 500 and 400HD contain enough material for 800 lanes GeneScan 1000 and 2500 contain enough material for 400 lanes GeneScan 500XL contains enough material for 1600 lanes Loading buffer is included 401735 GeneScan 350 ROX 401736 GeneScan 350 TAMRA 402985 GeneScan 400HD ROX 401734 GeneScan 500 ROX 401733 GeneScan 500 TAMRA 403040 GeneScan 500XL TAMRA 403039 GeneScan 500XL ROX 401098 GeneScan 1000 ROX 401100 GeneScan 2500 ROX 401545 GeneScan 2500 TAMRA 401144 Loading Buffer Fluorescent dNTPs For fluorescent labeling of DNA during PCR amplification C 8 Parts and Accessories 401894 F JdUT
69. WTR plates 4 25 29 1 polyacrylamide 1200 scans hr 4 75 Long Ranger Singel 750 900 5 25 PAGE PLUS Note The products required to make the gels presented in this appendix are not manufactured by Applied Biosystems The quality assurance of these products is regulated by the respective suppliers A 2 Gel Recipes Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Sequencing Chemistries Released in 1997 New Chemistries During 1997 three new sequencing chemistries were released Chemistry Part Numbers ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase FS 403044 for the 100 reaction kit 1000 reaction kit 403045 for the ABI PRISM BigDye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase FS 403051 for the 100 reaction kit 1000 reaction kit 403049 for the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase FS 4303149 for the 100 reaction kit 1000 reaction kit 4303150 for the When these new chemistries are used with the gel formulations listed under Recommendations for Longest Read Length on page A 2 read lengths commonly fall at the upper end of the ranges listed in the table The new chemistries produce longer read lengths because they have Greater signal to noise ratios Mo
70. a description of the prerun and its purpose IMPORTANT To avoid gel extrusion the electrophoresis voltage during a prerun should never exceed 1 kV Prerunning the gel is particularly important when performing a high speed 2400 scans hour run The gel must be at run temperature when the samples are loaded to ensure appropriate denaturation conditions To prerun the gel Step Action 1 If not already specified in the Run window open the PreRun pop up menu and select the appropriate PreRun module 2 Optional recommended when using a square tooth comb and if you are a new user Follow these steps to stain the surface of the gel Staining will help you visualize the wells and make sample loading easier a Load approximately 25 50 uL of formamide blue dextran loading solution into a pipet b Starting at one end of the wells slowly drag and release the formamide blue dextran loading solution across the tops of the wells CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Wear appropriate protective eyewear clothing and gloves Obtain a copy of the MSDS from the manufacturer 3 Close the front panel of the instrument Instrument Operation 3 35 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To prerun the gel continued Step Action 4 Click PreRun The Scan window
71. ae AS 8 14 Computer Maintenance Recommendations 0 0 0 0 cee cece eee eee ee 8 17 Restarting the Computer 0 2 ee cette ent E ene 8 18 Rebuilding the DesktOp lt ngs 40050 444 swede nee da yer ee eee s ould ca bede whee PAS 8 18 Deleting Data Files pepa Sates Sad Sark Gc ads eA ee ESN ares Mee 8 18 Optimizing the Hard Disk 0 0 0 eect ene ene 8 19 Backing Up Important FileS ies ccc sate ages bee aE ae be ye ea ee ee gd 8 20 SAM ea stint gp dee ee Md Me RE Soy ned Nae N RGSS eS sm 8 21 Norton UGS 05 2 scan gaciod tp hese dled ween nae ew eee dene eew eee Pee aoe Be 8 22 Archiving Data from Runs 1 2 ee ete E a e ne neee 8 23 Reinstalling ABI PRISM 377 Software 0 0 eect eee 8 25 Downloading Data Collection Software from Our Website 00 00 0005 8 26 Adding Non Applied Biosystems Software 0 0 0c cece eee eee 8 28 Systema File so 4 cdc edeas Helen tea E Sate hee ae ee oe eee dane 8 29 9 Data Collection Software 0 6 ccc ccc eee w eens Jad Chapter Contents siguerian weed ee de Se RG Sa dle ee 9 1 System Software Overview 00 ee cece nett e ence ene ees 9 2 Data Collection Software Files and Folders 0 0 cee cece eee 9 3 Setting Folder Location Preferences 0 0 cece ccc eee eee eens 9 5 Data Flow Between the Computer and Instrument 0 0 0 0c eee eee ee eee 9 7 Launching the Data Collection Software Program 0 00 c e
72. and can be viewed in the annotative view of the sample file This is useful for instrument identification particularly if more than one instrument is being operated See Using Calibration File Make and Send in Chapter 4 for more information CCD Gain Typically does not require adjustment When running dRhodamine terminators on an ABI PRISM 377 with XL Upgrade we recommend increasing the gain to 4 The default setting is 2 Instrument Operation 3 49 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 50 Instrument Operation Function Name Description CCD Offset Used to move baselines and signals up and down the scale in the Scan window and in the recorded data CCD Offset can be useful if baselines look unusually high or low Be aware that baselines normally drop over the course of a run Therefore the lowest baseline should be set high enough so that it does not drop below zero by the end of the run We recommend setting the lowest baseline at the beginning of the run between 300 and 500 points External Cooler On Relay 4 Used to manually control power to an external water bath This function gives you control of the internal system pump from the rear of the instrument at relay 4 Manual control is not required if the recommended external water bath and chiller modules are used Refer to Appendix B Subambient Temperature Operation
73. and in Chapter 9 and to step 5 of the process listed above A description of the plate check prerun and run are provided on pages 1 15 through 1 17 Configuring the software is described below The Data Collection ABI PRISM Data Collection Software is the interface between the instrument and the Software computer It performs the following functions Controls the instrument by sending it commands that are contained in files called modules Collects raw data from the instrument and stores it in a gel file Figure 1 1 on page 1 17 described on page 1 13 Transfers data automatically to either the sequencing or GeneScan analysis software for automatic data analysis at the end of a run software must be configured for this to occur Modules contain the various settings electrophoresis voltage gel temperature etc required for instrument operation Two sets of modules are included Standard modules provide gel temperature control from 10 C above ambient to a maximum of 60 C Chiller modules used when an external cold water bath is attached to the instrument and temperatures below 10 C above ambient are required There are three types of standard and chiller modules plate check prerun and run Prerun and run modules are specific for either sequencing or GeneScan applications Configuring the software for a run means setting up a sample sheet and a run sheet There are two types of sample and run sheets one for se
74. and run sheets both require the selection of additional information for their completion Refer to the following for more information About Sample Sheets for Sequencing Applications on page 9 14 or About Sample Sheets for GeneScan Applications on page 9 20 About Run Sheets on page 9 28 Modules on page 9 42 Output Files File Type Location Description The following files are created automatically by data collection software for each run Gel Log Run Contains the data collected during a run Gel files are typically All three files are located in an Individual Run folder inside the Runs folder inside the ABI PRISM 377 folder very large 20 70 MB The default naming convention used for gel files is Gel File lt date gt lt time gt Contains a date and time stamped comprehensive history of actions taken instrument and computer conditions and any errors received during a run The default naming convention used for log files is Log This file is a copy of the run sheet prepared for the run The run sheet is also an input file The default naming convention used for run files is Run File lt date gt lt time gt Locations valid only if folder location preferences are not changed See Setting Folder Location Preferences on page 9 5 and Chapter 5 Setting Preferences for more information Temporary Files Software creates a number of temporary files in the
75. arrow keys Press Tab to move to the next field to the right Press Return to move to the next field down To apply the same parameter to all fields in a column Step Action 1 Select the parameter in the top field of the column 2 Click the column title to select the entire column 3 Open Edit menu and choose Fill Down To copy information from one field to another Step Action 1 Click the field to be copied 2 Open the Edit menu and choose Copy 3 Click the new field 4 Open the Edit menu and choose Paste To copy an entire row of information to another row Step Action 1 Click the lane number in the left most column to select the entire row 2 Open the Edit menu and choose Copy 3 Click the lane number of the row you wish to copy into 4 Open the Edit menu and choose Paste Moving Information To move information to a different lane Step Action 1 Hold down the Option key and click the lane number of the row containing the information you want to move 2 Holding down Option key and the mouse button drag the lane number to another lane number When you release the mouse button the sample information appears in the new location 9 26 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Importing and Exporting Sample Sheet Informatio
76. at Name of new matrix file Points Update File Instrument Comment Dye Primer Matrix Tag Terminator Matrix T Terminator Matrix 3 Specify the sample file to be used for each standard as follows a Click the C button b Inthe directory dialog box that appears select the file that contains the data from the C standard then choose Open c Continue this selection process for the A G and T standards by clicking the A button to select the A standard and so on Note A separate matrix must be generated for each chemistry Fluorescein Rhodamine Box Dye Primer Matrix Taq Terminator Matrix T7 Terminator Matrix C FAM ROX not used As JOE R6G not used G TAMRA R110 not used Tes ROX TAMRA not used 7 8 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To make an instrument file for virtual filter set A continued Step Action 4 In each Start at text box leave the default as is or enter a different value Note The defaults of 2000 for start point and 1500 for data points to be used for the matrix are almost always appropriate Refer to the ABI PRism DNA Sequencing Analysis Software User s Manual for information on how to determine values for the Start at and Points boxes In the Points text box leave the default va
77. b Click OK The computer makes the matrix When finished a dialog window appears with the message Make matrix successfully completed c Click OK 7 If the computer is unable to make a matrix examine the raw data again in the Sequencing Analysis software If you used the default values then select new start points as directed in steps 8 and 9 If many peaks are off scale dilute the matrix standards and rerun them 7 12 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To make the Dye Primer Matrix continued Step Action 8 If the matrix cannot be made with the default values proceed with steps a b andc below a Inthe Sequencing Analysis software open a matrix standard sample and examine the raw data An example is shown below Select a starting point where there are no peaks and the baseline is flat c Select a number of data points to analyze such that no peaks in the range are off scale i e above 4000 relative fluorescence units RFU and that the baseline at the end of the range is flat A typical number of data points is 1500 a 23edROH matrix std El 1920 2000 2080 2160 2240 1358_ 1142_ 926_ Fig i 494_ 278 AEP ME O Repeat step 8 for each matrix standard sample Record the results for later use IMPORTANT The number of d
78. be on scale and the dye of interest should have a value of at least 200 2 Check for any data anomalies such as an unstable baseline Rerun samples that have an unstable baseline 6 6 Making Matrix Files for GeneScan Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Step Action Select a starting point for the matrix data as shown below The starting point for matrix data should be slightly beyond the point where the primer peak falls back to the baseline approximately 2950 scans in this example a A3ened FE 2700 3000 3300 3600 3900 4200 start point Choose a stop point such that at least three matrix standard peaks will be within the range analyzed Generating the Matrix File To generate the matrix file Step Action 1 Choose New from the File menu 2 Click the Matrix icon The Make New Matrix dialog box is displayed Make New Matrix Select the Matrix Standard Sample Files No File Selected for B Data Start At s No File Selected for G Data Start At fp fp x No File Selected for Y Data Start At fo po No File Selected for R Data Start At Points 100000 Click the B G Y and R buttons to choose the standard sample files Choose the sample file representing blue dye for B green dye for G etc
79. both plates in the lower buffer chamber electrical contact is lost and the instrument shuts down automatically to prevent arcing a luminous low voltage high current electrical discharge that can severely damage the instrument Bsa r ca Ca Cs Instrument shuts down automatically if a a buffer level drops below the notch at the D CS top of the front plate or below the bottom of the plates during a run ec ce LCs C 5 Carefully fill the lower buffer chamber with 1X TBE buffer to the top edge of the overflow dam Do not overfill Note Be careful not to splash buffer up onto the back of the rear plate Buffer on the plate will dry during the run resulting in a green haze on the gel image 6 Proceed to Installing the Front Heat Transfer Plate on page 3 18 Instrument Operation 3 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Installing the Front Heat Transfer Plate Install the Front To install the front heat transfer plate Heat Transfer Plate Step Action 1 If thisisa Then 48 cm run use of the front heat transfer plate is optional and is not necessary for good data Proceed to step 2 and install the plate or proceed to Setting Up the Software for a Run
80. by a drying cycle After some experimentation you may be able to reduce the rinse time Do not use a detergent Avoid excessive handling of dry plates with ungloved hands Detergents Alconox most major laboratory suppliers Multiterge VWR Scientific Products P N 34171 010 WARNING CHEMICAL HAZARD Acrylamide and bisacrylamide are poisons neurotoxins irritants carcinogens and possible teratogens Acrylamide and bisacrylamide sublime the solids release toxic vapor and are harmful if swallowed inhaled or absorbed through the skin Effects are cumulative When handling always wear protective equipment lab coat safety glasses and chemical resistant gloves and use in a well ventilated area On a routine basis thoroughly clean surfaces subject to contamination Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Procedure If a dishwasher is not available follow this procedure to manually clean glass plates Step Action 1 Put on a pair of gloves If necessary wash gloves to remove talc powder IMPORTANT Always wear gloves when handling glass plates to avoid skin contact with the gel and to avoid contaminating clean glass plates If the plates Then contain a gel a Gently pry the plates apart starting at the bottom of the plates Do not pry plates apart at the ears Be careful not to chip the plates pr Remove the comb and s
81. communicating but the instrument does not respond appropriately the firmware image may be corrupted Download a new copy of the firmware using Cold Boot Instrument Procedure To cold boot the instrument Step Action 1 Select Manual Control from the Window menu in the data collection software program Open the Fxn Name pop up menu Select Cold Boot Instrument and click Execute The current firmware image is erased from memory and the status lights on the front of the instrument change from green ready to flashing yellow Open the File menu and select Quit Launch the data collection software program The firmware is automatically downloaded to the instrument Note If the following dialog box is displayed see Locating the Firmware Image File on page 4 24 amp Firmware Image Y D ABI Prism 377 Firmware Eject Desktop Cancel Where Is ABI Prism 37 Firmware Troubleshooting 4 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Locating the Firmware Image File Location of the The firmware required to run the instrument is stored in memory on the instrument A Firmware File copy of the firmware is also included as part of the data collection software and is stored in the ABI PRISM 377 folder The location of the firmware on the computer must be specified as one of several Folder Location Preferen
82. corresponding run folder along with the Gel Log and run sheet files lt File Name gt seq Created by ABI PRISM DNA Sequencing Analysis Software only these files show the Files base letter sequence of the data The files can also contain a header and are stored in the corresponding run folder along with the Gel Log run sheet and sample files seq files can be opened from word processing programs and printed They can also be saved in several formats that can be read by other software programs The Preferences dialog box in the sequencing analysis software program allows you to choose a file format 9 60 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com ABI PRism 377 DNA Sequencer Appendix A Gel Recipes Quick Reference Guide BSS BibEystems Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Gel Recipes Appendix Contents In this Appendix The following information is provided in this appendix Topic See page List of Gel Recipes and Recommendations A 2 Sequencing Chemistries Released in 1997 A 3 Factors that Affect Gel Quality and Read Lengths A 4 Chemical Abbreviations Used A 9 Preparing TBE Buffer A 9 Deionizing Formamide A 10 2
83. default values Your numbers may vary Make Matrix 23edROH matrix std Start at 17 dR66 matrix std Start at 6 21 dR110 matrix std Start at 19 dTAMRA matrix std Start at Points dRhod_BigDye Instrument Comment Dye Primer Matrix Taq Terminator Matrix T Terminator Matrix 7 a Click OK The computer makes the matrix When finished a dialog window appears with the message Make matrix successfully completed b Click OK 7 14 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Making the T7 To make the T7 Terminator Matrix Terminator Matrix Step Action 1 In the Data Utility application choose Make Matrix from the Utilities menu The Make Matrix dialog box appears In the Make Matrix dialog box click the T7 Terminator Matrix button at the lower left Click on the box for each nucleotide base and enter the data file that corresponds to the correct matrix standard as shown in the table below note the order of the matrix standard files T7 Terminator Box Matrix dR6G dTAMRA dROX dR110 70 gt O Enter the same numbers for each matrix standard sample in the Start at and Points boxes as were used in the Dye Primer Matrix and Taq Terminator Matrix Click Update File A dialog window appears Choose dRhod_BigDye from the ABI folder
84. green lines can be transposed or displayed on top of each other This is acceptable Watch the scan lines for approximately 30 seconds If Then the scan lines are relatively flat the plates are clean Figure 3 2 on page 3 12 a Click Cancel and terminate the Plate Check b Leave the run window open Proceed to Installing the Upper Buffer Chamber on page 3 15 peaks appear in the scan window the plates are dirty Proceed to Reclean Figure 3 2 on page 3 12 the Plates on page 3 13 the green line is higher than the red the plates or gel are contaminated with a and black lines large amount of fluorescent material Proceed to Reclean the Plates on page 3 13 Instrument Operation 3 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 12 Instrument Operation Ee scan Window SSS Scan Window X 386 F x 82 Y 2356 Freeze Updates Current Scan Number 16 Y 6600 O Freeze Updates Current can Number 8 81924 m anon at 61444 40364 el a jannaa N 2048 ag a PE NT nee re ee n mi he e re So fast o T z c Clean plates Dirty plates Figure 3 2 Scan windows during the plate check showing clean and dirty plates The typical order of colors for the scan lines from top to bottom is red black blue and green Artisan Technology Group
85. indicates less than 70 C Note The error message Heat plate temperature exceeds 70 C is triggered by either the plate sensors or heater sensor If the flow switch sticks in the flowing position due to calcium deposits the pump shuts down but the heater continues to operate in an attempt to raise the temperature of the front heat transfer plate When the temperature of the heater exceeds 70 C the error message is displayed Flush the system with a mild acid solution to remove calcium deposits The cleaning solution and procedure are listed below Refill the water reservoir with a solution of deionized water and 5 0 antifreeze Refer to Refilling the Water Reservoir in Chapter 8 System Maintenance for more information Calcium deposits can usually be removed by flushing the system with a mild acid solution Commercially available de scaling solutions for removing deposits in coffee pots or hot water systems are recommended Follow all precautions on the label when working with these solutions If necessary use a 3 0 by volume nitric acid solution WARNING Donotuse nitric acid with a higher concentration Full strength nitric acid is 15 99N and should be diluted for use in this procedure WARNING CHEMICAL HAZARD Nitric acid fuming is an extremely corrosive oxidant 3 nitric acid should be prepared by trained personnel only Do not inhale vapor Work in a well ventilated area in a fume hood and wear re
86. is displayed 5 Open the Window menu and select Status to display the Status window Time remaining for the module being executed Instrument State Running Laser Power m Running Electrophoresis Power On Door me Closed Time Remaining 00 58 29 f Total 01 00 00 4 Total amount of time Electrophoresis Electrophoresis Electrophoresis Gel Laser the module will run Yoltage kY Current mA Power W Temperature C Power mW Green arrow indicates actual reading from instrument Gray box indicates value set by the module Electrophoresis voltage gel temperature and laser power are setpoints gt gt Electrophoresis current and power are limits Prerun the gel only until run temperature gel temperature in the Status window is reached approximately 15 25 minutes The default duration of the prerun module may be longer so watch the Time Remaining in the Status window shown above Note Prerunning the gel longer than it takes to reach run temperature can cause it to swell up around the comb If this occurs the samples may not run straight in the gel and resolution will be adversely affected The surface of the gel where the samples are loaded should be as level as possible If contaminants are present in the gel and you have elected to skip contaminated lanes when loading samples a Click on the Scan window to activate it
87. izy cm Gel s Matrix File lt none gt v Operator Lo o o v Lane Sample Sample Name Sample File Name Matrix File Auto Analysis Parameters Size Standard Data Collection Software 9 29 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Run sheets for GeneScan analysis applications contain the following information Parameter Description Plate Check Module File containing the instrument settings for the plate check PreRun Module File containing the instrument settings for the prerun Run Module File containing the instrument settings for the run Collect time The length of time in hours data will be collected Sample Sheet The sample sheet prepared for the run When selected the information from the sample sheet is imported to the sample information fields on the run sheet Well to Read Distance The well to read distance of the glass plates in cm Gel s Matrix File A mathematical matrix used to compensate for the spectral overlap that occurs between the dyes used together as a set Matrices are dye set instrument and run condition dependent As such matrices must be remade when any of these conditions change A matrix file must be selected for data to be analyzed automatically For more information refer to About Automatic Data Analysis on page 9 38 Chapter 6
88. lanes Full Scan 24 32 36 lanes XL Scan 48 50 64 66 lanes XL Scan 48 64 lanes 96 Lane Scan 96 lanes 96 Lane Scan 96 lanes Note If this is an XL or 96 lane upgrade instrument the appropriate value for the Run Mode field is selected automatically when this parameter is set The run sheet for the standard ABI PRISM 377 instrument does not have a Run Mode field Select the IMPORTANT Select the number of lanes before selecting the sample sheet If the default Sample Sheet umber of lanes is less than the number of samples the sample information on the run sheet will be truncated For example if you entered 36 samples on the sample sheet but the default number of lanes on the run sheet is 24 only the information for the first 24 samples will be imported to the run sheet if the sample sheet is selected before the number of lanes is changed to 36 To select a sample sheet Step Action 1 Open the sample sheet pop up menu and select the sample sheet prepared for this run Once selected the information on the sample sheet is imported to the run sheet IMPORTANT If the pop up menu lists lt none gt only software cannot find the folder that contains the sample sheets To correct this see Setting Folder Location Preferences on page 3 51 Instrument Operation 3 29 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Step Action 2 T
89. lower high voltage connection 4 Fit the cassette up against the rear heat transfer plate between the four clamps in the chamber as shown below Turn the clamps to lock the cassette in place IMPORTANT Do not touch the read region of the plates see page 3 47 Clamps locking cassette in place GRO355 A Electrode cable from Lower buffer lower buffer chamber chamber Instrument Operation 3 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To install the gel cassette and lower buffer chamber continued Step Action 5 Carefully check cassette installation The positioning pins in the chamber Figure 3 1 on page 3 8 must be touching the spacers through the two holes on the back of the cassette The pins should be visible through the glass Repeat the installation if necessary IMPORTANT Proper installation is critical to ensure good results The rear heat transfer plate is spring loaded and is designed to apply even pressure to the glass plates If the cassette is not properly installed against the positioning pins instrument sensitivity will be greatly reduced and experimental results will be poor Close the front panel of the instrument and proceed to Performing a Plate Check on page 3 11
90. mm of each end Wearing latex gloves apply a 1 16 1 5 mm diameter bead of sealant into the center of the groove Apply a consistent amount throughout the groove Place the gasket in the groove Starting from one side of the chamber use your index finger to gently press the gasket into the groove with a rolling motion IMPORTANT Do not use excessive force to push the gasket into the groove Excessive force or stretching the center portion of the gasket can distort the material and adversely affect sealing Remove excess sealant with a Kimwipe Be especially careful that no sealant is left on the surface of the gasket that will contact the glass plate Stretch the ends of the gasket to the ends of the groove if necessary and hold the ends in place for 10 seconds CAUTION Never stretch the center portion of the gasket 8 8 System Maintenance Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To install the new gasket continued Step Action 7 Place the scrap glass or plastic plate on top of the gasket making sure the plate sits flat IMPORTANT Use scrap glass only Do not use glass plates that are also used for gels Residual sealant will ruin gel plates Press gently and evenly on the plate with your fingertips and hold for 15 seconds Visually verify that there are no gaps between the plate and the gas
91. of the overflow dam Do not overfill No communication between instrument and computer Instrument memory cleared due to a power outage Corrupted firmware on instrument Cable is loose Perform a single reset See Locating the Firmware Image File on page 4 24 If this does not solve the problem perform a total reset See Performing a Total Reset on page 4 22 Flat scan lines or no scan lines Instrument memory cleared due to a power outage Corrupted firmware on instrument Perform a single reset See Locating the Firmware Image File on page 4 24 Error message EP Voltage Deviation Exceeds Tolerance The EP voltage deviated outside the tolerance range Instrument operation is paused Call service Error message No EP Current Detected Concentration of TBE buffer in buffer chambers differs from the concentration in the gel All should be 1X TBE TBE buffer concentration must be the same in the gel and both buffer chambers Remake the gel and buffer chamber solutions so the TBE buffer concentration is the same in each 4 6 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Observation Possible Causes Recommended Actions Error message Warning Possible Plate P43 J43 Thermistor Open Short Circuit Warning Plate Out Thermistor P43 J43 Open Short Circ
92. part numbers C 7 Instrument menu about instrument operation cleaning glass plates 3 4 to 3 5 cleaning up after run 3 45 filling buffer chambers 3 16 to 3 17 installing front heat transfer plate 3 18 installing gel cassette 3 8 to 3 10 installing lower buffer 9 12 chamber 3 8 to 3 10 installing upper buffer chamber 3 15 loading gel into cassette 3 6 to 3 7 loading samples 3 35 to 3 39 manually controlling the instrument 3 48 to 3 50 materials required but not supplied 3 2 new user recommendations 1 3 performing plate check 3 11 to 3 13 performing prerun and loading samples 3 35 to 3 39 preparing 1X TBE buffer solution 3 3 preparing GeneScan sample sheet 3 24 to 3 27 preparing loading solution 3 3 preparing run sheet 3 28 to 3 34 preparing sequencing sample sheet 3 19 to 3 23 read region description of setting folder location preferences 3 51 to 3 53 setting up the software an overview 3 19 skipping lanes determining which to skip 3 14 starting and monitoring the run 3 40 to 3 43 summary of procedures 3 2 theory of operation 1 13 3 47 troubleshooting guide 4 6 to 4 9 viewing the Log file 3 44 instrument status Electrophoresis History window 9 56 Status window 9 53 internet address Documents on Demand 1 10 part number updates C 1 1 9 to K keyboard commands shortcuts KOH wash performing 4 30 9 9 L lane tracking affects of air bubbles A 8 laser safety bar 3 18 safety warn
93. pitch eight channel loaders C 5 to C 6 Symantec AntiVirus program about 8 21 system file about 8 29 system software 9 2 reinstalling 8 25 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com T tab delimited text importing or exporting 9 27 technical support 1 6 to 1 10 e mail address 1 6 internet address 1 9 telephone fax 1 6 to 1 9 technical updates to user s manual 1 5 TEMED effect of changing concentration A 8 oxidized form A 6 safety warning A 6 A 23 storing A 23 temperature how affects gel quality A 8 temperature run 3 35 ensuring appropriate denaturation conditions 1 16 temporary files created in System folder 9 8 terminators dye BigDye 7 4 dRhodamine 7 4 Rhodamine 7 4 theory of operation 1 13 total reset 4 22 tracking affects of air bubbles A 8 Tracker program red data requirement 7 7 using the GelDoc utility 1 19 Tris contaminants reagent purity and associated problems A 6 Tris borate EDTA chemical warning 3 3 troubleshooting amplification 4 16 CCD camera aligning 4 25 to 4 26 CCD pixel position Calibration File Make and Send 4 27 to 4 28 CCD pixel position value 4 25 cold boot procedure 4 23 coolant system clogs 4 32 Data Collection software 4 10 firmware 4 21 gasket marks on glass plates 4 31 gel and gel image 4 3 to 4 4 gel extrusion 4 29 if preferences file becomes corrupt 5 4 instrument 4 6 to 4 9 log file error messages 4 20 quick
94. platform e g two empty pipet tip boxes Glass plates front and rear one set Spacers two 0 2 mm gt gt gt gt ad Syringe 60 cc Preparing the Gel Prepare the gel solution now See Selecting a Gel Formulation on page 2 2 for Solution guidelines Gel recipes are listed in Appendix A Gel Recipes IMPORTANT The polymerizing reagents ammonium persulfate and TEMED are the catalysts that initiate polymerization Once these reagents are added to the gel solution you must work quickly to pour the gel Polymerization can occur within 15 minutes If desired you can prepare the solution up to the point of adding the polymerizing reagents then 1 prepare the work area and glass plates 2 add the polymerizing reagents to the gel solution and 3 pour the gel Preparing the Poria a and To prepare the work area and glass plates Glass Plates prep 9 p Step Action 1 Prepare a raised platform that is stable and level For example two empty pipet tip boxes work well 2 Check the inside surface of both plates for water droplets dust lint or anything else that might fluoresce or scatter light Clean the plates with a damp Kimwipe if necessary 3 Place the rear plate on the platform Place absorbent paper towels under the plate to catch spills or overflow 5 Check the spacers and comb If necessary wipe them off with a damp Kimwipe 2 20 Pouring Gels Artisan Technology Gr
95. preceding protocol in two places Step 2 on page A 16 Do not add urea Step 3 on page A 16 Add the following 8 1 mL 50 Long Ranger gel solution concentrate 1 25 g glycerol 5 0 mL 10X TBE Gel Recipes A 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com MDE Gel Protocol for SSCP Action Instructions for preparing glass plates and two gel pouring methods are located in Chapter 2 Pouring Gels Glass plates and all other gel pouring equipment must be ready for use prior to adding the polymerizing reagents to the gel solution Combine the following 10 0 mL 2X Mutation Detection Gel Solution MDE 4 0 mL 10X TBE 2 0g glycerol WARNING MDE Mutation Detection Gel Solution contains Acrylamide Acrylamide is a poison neurotoxin irritant carcinogen and possible teratogen Acrylamide sublimes the solid releases toxic vapor and is harmful if swallowed inhaled or absorbed through the skin Effects are cumulative When handling always wear protective equipment lab coat safety glasses and chemical resistant gloves and use in a well ventilated area On a routine basis thoroughly clean surfaces subject to contamination Add sufficient distilled deionized water to bring the total volume to 40 0 mL Mix well to obtain a homogenous solution Note This step is critical because glycerol is viscous and it is difficult to obtain a homogen
96. price of this instrument and corresponds to the up front fee component of a license under process claims of U S Patent Nos 5 821 058 and 5 332 666 and under all process claims for DNA sequence and fragment analysis of U S patents now or hereafter owned or licensable by Applied Biosystems for which an Authorization is required and under corresponding process claims in foreign counterparts of the foregoing for which an Authorizaton is required The running royalty component of licenses may be purchased from Applied Biosystems or obtained by using Authorized reagents purchased from Authorized suppliers in accordance with the label rights accompanying such reagents Purchase of this instrument does not itself convey to the purchaser a complete license or right to perform the above processes This instrument is also licensed under U S Patent No 5 171 534 and apparatus and system claims in foreign counterparts thereof No rights are granted expressly by implication or by estoppel under composition claims or under other process or system claims owned or licensable by Applied Biosystems For more information regarding licenses please contact the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 ABI PRISM the ABI PRISM design Applied Biosystems GeneScan Genotyper INHERIT MicroAmp and Sequence Navigator are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countr
97. problem with a specific INIT follow this procedure To turn off specific INITs Step Action 1 Open the Extensions Manager window as follows a Open the Apple menu b Select Control Panels and open the control panels menu c Select Extensions Manager 2 Click the suspect INIT to remove the checkmark and turn it off 3 Open the Special menu and select Restart to restart the Macintosh 4 Try running the system without the INIT to see if this solves the problem To turn off all INITs Step Action 1 Restart the Macintosh while holding down the Shift key to turn off all INITs To turn all INITs back on Step Action 1 Restart the computer Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com System File Overview Only one file named System is required to operate the Macintosh computer The System file is located in the System Folder on the hard disk and is essential for all operations on the Macintosh The System folder and file are set up on the computer during system installation Other software programs sold by Applied Biosystems that can be used on the ABI PRISM 377 DNA Sequencer do not include a System file However applications sold by other manufacturers might include a System file We strongly recommend that you do not install other System files if additional software is installed See
98. proceed to Chapter 3 Instrument Operation for step by step instructions on instrument set up and operation Note Chapters 2 and 3 and Appendix A are bound separately for your convenience They can be removed from the binder and used separately as quick reference guides for preparing gels and using the instrument 1 2 About This Instrument Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com What to Do If You Are a New User Safety Information The Macintosh Computer What to Read Before using the instrument read the safety information located on page 1 4 and in the ABI PRISM 377 Site Preparation and Safety Guide P N 903393 This manual is written with the assumption that you know how to operate a Macintosh computer If you are not familiar with this computer refer to the Macintosh documentation shipped with this system for specific operating information We recommend reading ABI PRISM 377 DNA Sequencer System Components on page 1 11 Provides an overview of system hardware and software Theory of Operation on page 1 13 Describes electrophoresis and how data is collected Using the Instrument on page 1 14 Provides an overview of the steps involved in using the instrument Introduces terminology specific to system operation Chapter 2 Pouring Gels and Appendix A Gel Recipes if you are preparing your own gels
99. settings 9 43 to 9 44 Mutation Detection gel See MDE gel N new user recommendations 1 3 nitric acid wash 4 32 nitric acid waste explosion warning 4 32 Norton Utilities about 8 22 using Disk Doctor 4 20 Note user attention word defined 1 5 O opening files 9 57 operating the instrument See instrument operation organic solvents and glass plates 2 7 Index 6 overlay strips part numbers C 3 using 3 5 P PAGE PLUS gels gel recipes and recommendations A 2 ingredients and run conditions A 13 protocol A 14 part numbers buffer chambers C 3 combs and overlay strips C 3 electrode assemblies C 4 electrophoresis cables C 4 fragment kits and reagents C 8 to C 11 front heat transfer plate C 4 gasket kits for upper buffer chambers C 3 gel cassette and pouring fixture C 3 glass plates spacer and clamps C 2 two pitch eight channel loader suppliers C 5 to C 6 updates via the web C 1 user s manuals C 7 PCR Single stranded Conformation Polymorphism See SSCP pipetting ergonomic warning and suggestions 1 4 3 37 plate check commands to perform 9 39 to 9 41 electrical shock hazard warning 3 11 how to perform 3 11 purpose of 1 15 to 1 16 plates cleaning cleaning by hand 2 6 to 2 7 recommended dishwashers 2 8 using adishwasher 2 6 cleaning before loading gel 3 4 to 3 5 gasket mark 2 5 identifying front and back 2 5 part numbers C 2 read region description of 3 47 using new glass plates 2 5 Polyacrylamide ge
100. sheet specific to your application sequencing or GeneScan The location of instructions for preparing sample and run sheets are listed in the following table Application Type of Sheet Location of Instructions Sequencing Sample sheet page 3 19 Run sheet page 3 28 GeneScan Sample Sheet page 3 24 Run sheet page 3 28 Preparing a Sequencing Sample Sheet Save Time by Setting Sample Sheet Preferences Project Names and BioLIMS IMPORTANT Do not mix sequence and GeneScan analysis samples on the same sample sheet or in the same run If the same type of run is performed repeatedly on the same instrument you can reduce the time spent setting up sample sheets by setting sequencing sample sheet default preferences Setting sample sheet preferences means changing the default value of certain fields on the sample sheet template to the values used most often The following fields on sequencing sample sheets can be set as preferences DyeSet Primer Instrument File Once these preferences have been set the preferred values appear automatically on each new sequence sample sheet Preferences can be changed as often as necessary either by setting new preference values or by opening the pop up menus and manually selecting new values Instructions for setting sequencing sample sheet default preferences are located in Chapter 5 Setting Preferences BioLIMS and Setting Preferences The Project Name field o
101. standard modules are the ones displayed in the B 2 Subambient Temperature Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com pop up menus on run sheets Chiller modules are stored in a folder called Chiller Modules You must either move the chiller modules to the Modules folder or change the Folder Locations Preference for Modules to the Chiller Modules folder for the software to access the chiller modules Refer to Installing Chiller Modules on page 9 44 in Chapter 9 Data Collection Software and Folder Location Preferences in Chapter 5 Setting Preferences for instructions on how to access the chiller modules Requirements for The following table lists the requirements for standard and SSCP operation of the Specific Run instrument Conditions Data Collection Thermistor Run Conditions Software Version ohms External Bath gt 10 C above ambient to 60 C 1 1 or 2 1 100 k not required SSCP from 22 C to ambient 10 C 2 1 100 k required SSCP at lt 22 C 2 1 30 k required How the External Water Bath Functions Disengages Internal The instrument configuration with the external water bath attached is similar to Pump System Figure 1 when viewed from the back Instructions in the chiller modules disengage the instrument s internal pump system and switch to the external water bath When the external valve is turned so the arrow p
102. the clear brace as follows a Slide the pegs of the brace up against the cassette clamps at the top of the plates b Fit the ears of the brace over the ears of the front glass plate IMPORTANT The brace must cover the ears of the front plate to prevent leakage and hold the comb in place Ear 4 Peg of clear brace positioned against clamp before clamp is closed j Insert the flat edge of the comb between the plates as far as it will go Lock the clear brace in position by turning the appropriate two clamps Test the tightness of the comb between the plates by carefully pulling on the tips of the teeth If either side or both sides of the comb can be pulled out from between the plates tighten the screws on the underside of the black brace Note Testing the fit of the comb between the plates at this point in the procedure is more efficient than waiting until you are ready to pour the gel You may not have enough time to make the appropriate adjustments if you perform this test after the polymerizing reagents are added to the gel solution IMPORTANT Keep the braces locked in place throughout the remainder of this procedure Check the luer fitting in the gel injection fixture Finger tighten the fitting if it is loose If the fixture is still wet from previous use briefly blow air through the luer fitting to expel any trapped moisture A
103. the list of folders Do not click Open 5 If Then you are finished click OK to save your changes or Cancel to quit without making any changes you wish to change more preferences open the Page pop up menu and select another Preference Instrument Operation 3 53 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Index Numerics 10X TBE buffer solution 3 2 3 3 1X TBE buffer solution preparing 3 3 377 instrument See ABI Prism 377 A ABI settings folder location and description of 3 52 ABI Prism 377 changing settings in real time 3 48 instrument operation cleaning the glass plates 3 4 to 3 5 cleaning up after run 3 45 filling buffer chambers 3 16 to 3 17 installing front heat transfer plate 3 18 installing lower buffer chamber 3 9 installing upper buffer chamber 3 15 loading gel into cassette 3 6 to 3 7 loading samples 3 37 manually controlling the instrument 3 48 to 3 50 materials required but not supplied 3 2 performing aprerun 3 35 to 3 39 performing plate check 3 11 to 3 13 preparing 1X TBE buffer solution 3 3 preparing arun sheet 3 28 to 3 34 preparing GeneScan sample sheet 3 24 to 3 27 preparing loading solution 3 3 preparing sequencing sample sheet 3 19 to 3 23 read region description of 3 47 removing gel ca
104. the run Instrument and Macintosh errors Log files are stored in the Run folder To view the Log file open the Windows menu and select Log Refer to Chapter 9 Data Collection Software for more information on this window gt 3 2 99 3 49 37 PM gt 3 2 98 3 49 37 PM gt 3 2 98 3 49 42 PM gt 372798 3 49 43 PM 3 2 93 3 49 54 PM 342 98 3 50 59 PM gt 3 2 98 3 51 00 PM ABI Prism 377 96 Collection version 2 5b3 ABI Prism 377 96 Firmware version 2 2 0 Module sent Plate Check D 450 Run Started Instrument Paused Run Resumed Instrument Resumed 572 98 3 51 01 PM Message Starting scan gt 3 2 98 3 53 05 PM gt 3 2 98 3 53 08 PM 3 2 98 3 55 53 PM gt 3 2 98 3 55 53 PM Run Cancelled Number of Scans Processed O Module sent GS PR 36D 2400 480 Run Started 372 98 3 56 13 PM Message Starting scan 3 44 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Cleaning Up After the Run Cleaning Procedure To clean up after a run Step Action 1 Optional Turn the instrument off Open the front panel of the instrument Disconnect the upper and lower buffer chamber electrode cables 2 3 4 If the front heat transfer plate was used disconnect the grounding strap and water supply tubes Remove the front heat transfer plate by releasing the
105. the sample sheet data To make changes to the sample sheet after selecting it on the run sheet a Open the sample sheet by clicking the icon next to the sample sheet pop up menu on the run sheet Sample Sheet e Click this icon Make changes to the sample sheet Close and save the sample sheet Open the sample sheet pop up menu on the run sheet and select lt none gt ogos Open the sample sheet pop up menu on the run sheet and reselect the sample sheet You cannot make changes to the sample sheet while a module is running 9 32 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Selecting the Instrument or Gel s Matrix File For GeneScan runs in particular matrices are dye set instrument and run condition dependent As such matrices must be remade when any of these conditions change For more information refer to Chapter 6 Making Matrix Files for GeneScan and the ABI PRISM GeneScan Reference Guide P N 4303188 To select an instrument or gel matrix file If thisisa Then sequencing run open the Instrument File pop up menu and select the appropriate file If one does not yet exist leave the field set to lt none gt A file must be selected for automatic data analysis GeneScan run automatic data analysis open the Gel s Matrix File pop up menu and select the appropriate file If one do
106. to 3 7 loading samples 3 37 manually controlling the instrument 3 48 to 3 50 materials required but not supplied 3 2 performing a prerun 3 35 to 3 39 performing plate check 3 11 to 3 13 preparing 1X TBE buffer solution 3 3 preparing arun sheet 3 28 to 3 34 preparing GeneScan sample sheet 3 24 to 3 27 preparing loading solution 3 3 preparing sequencing sample sheet 3 19 to 3 23 read region description of 3 47 removing gel cassette 3 45 setting folder location preferences 3 51 to 3 53 setting up the software 3 19 skipping lanes determining which to skip 3 14 starting and monitoring the run 3 40 to 3 43 theory of operation 1 13 viewing the Log file 3 44 maintenance CCD pixel position value 8 14 to 8 16 cleaning accessories 8 3 to 8 4 recommendations 8 2 refilling water reservoir 8 5 replacing electrophoresis cables and electrodes 8 12 to 8 13 replacing upper buffer chamber gasket 8 6 to 8 11 part number updates C 1 reinstalling software 8 25 safety warnings 1 4 software 9 2 subambient temperature operation avoiding condensation B 7 hardware and software required B 2 to B 3 how external water bath works B 3 to B 4 installing external water bath B 5 performing a run B 6 purpose of external cooling system B 1 See Also SSCP summary of operation procedures 3 2 system components 1 11 to 1 12 hardware 1 11 models available 1 12 software 1 11 9 2 troubleshooting guide 4 6 to 4 9 viewing status 9 53 to 9
107. to the ABI Folder See Setting Folder Location Preferences on page 9 5 If the file is the same for all remaining samples click in the column heading to select the entire column open the Edit menu and select Fill Down Otherwise select the appropriate DyeSet Primer file for each sample individually Instrument File Battie Window Undo Pe ene ane Sample Sheet Cut Sequencing St Copy DyeSet Primer Click in this field to select Paste the entire column Any PrimD Sa gt Click these boxes to l Select All A l Fill Down 3D open pop up menus ig Set Scale ji P imi Show Clipboard z 7 Selecting the The instrument file must be the same for all the samples Instrument File To select the instrument file Step Action 1 Open the Instrument File pop up menu for the first sample and select the appropriate file You must select a file if you want the data to be analyzed automatically at the end of the run IMPORTANT If the pop up menu lists lt none gt only software cannot find the folder that contains the instrument files To correct this you must set the Settings Folder Preference to the ABI Folder See Setting Folder Location Preferences on page 9 5 Click in the column heading to select the entire column open the Edit menu and select Fill Down to enter the same instrument file for the remaining samples 9 18 Data Collection Software Artisan T
108. troubleshooting guide 4 19 sample names restrictions 3 20 3 24 9 14 9 17 sample sheet folder location and description of 3 51 sample sheets changing after selecting on run sheet 3 30 entering information 9 26 GeneScan applications about 9 20 to 9 21 preferences setting 5 12 preparing 9 22 to 9 25 importing exporting information 9 27 preparing forGeneScanrun 3 24 preparing for sequencing run 3 19 to 3 23 sequencing applications about 9 14 to 9 15 preparing 9 16 to 9 19 setting preferences 5 10 viewing 9 57 samples loading ergonomic warning and suggestions 3 37 performing prerun and loading samples 3 35 to 3 39 Index 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com saving automatically created files ensuring disk space 3 40 data from runs 8 23 to 8 24 files 9 57 sequence files 9 60 See Also archiving Scan window description of 3 41 9 55 use during Plate Check 3 11 Sequence Navigator software description of software sequencing chemistries A 3 sequencing software automatic data analysis 3 32 9 38 description of software instrument files adding replacing matrices 7 20 to 7 21 color guide for data display 7 4 to 7 5 DataUtility program description of 1 22 1 19 program 7 3 for Virtual Filter Set A 7 6 to 7 9 for Virtual Filter SetE 7 10to 7 15 making from a sample file 7 18 to 7 19 overview 7 2 storing an backing up 7 19 summary of c
109. up the drying process Compressed air propellants and organic solvents may leave fluorescent residues Pouring Gels 2 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Dishwasher The following dishwashers and plate rack are recommended for cleaning glass plates Recommendations Item P N Supplier s Lancer 1600 Dishwasher with Lancer 1600 UP Lancer USA Inc facility for drying 705 West Highway 434 Sequencing plate rack witha SPR 16 Longwood Florida 32750 50 plate capacity for Lancer Telephone 407 332 1855 1600 dishwasher Lancer UK Ltd 1 Pembroke Ave Waterbeach Cambridge CB5 9QR Telephone 44 01223 861665 Fax 44 01223 861990 Labconco Undercounter 15 352 801 Fisher Scientific SteamScrubber U S Headquarters Washer Dryer 585 Alpha Drive Pittsburgh Pennsylvania 15238 Customer Service 1 800 766 7000 Fax 1 800 926 1166 Internet http www fishersci com Cleaning Spacers WARNING CHEMICAL HAZARD Acrylamide and bisacrylamide are poisons and Combs neurotoxins irritants carcinogens and possible teratogens Acrylamide and bisacrylamide sublime the solids release toxic vapor and are harmful if swallowed inhaled or absorbed through the skin Effects are cumulative When handling always wear protective equipment lab coat safety glasses and chemical resistant gloves and use in a well ventilated area On a routine basis tho
110. www artisantg com CCD Pixel Position Value Overview The charge coupled device CCD pixel position value is a reference point for the Calibration File Make and Send alignment of the CCD camera with the laser beam The instrument is shipped with the correct CCD pixel position value stored in its memory When you start a run the Data Collection software checks for a value greater than zero If not found the following error message is displayed Could not complete your request because the CCD pixel position is incorrect Follow the procedures Location of the CCD Pixel Position Value and Checking and Entering the CCD Pixel Position Value on page 4 26 to locate and reenter the value Two functions Calibration File Make and Calibration File Send allow you to create a file that contains the CCD pixel position value and instrument serial number This file is called ABI 377 Calibrations and is stored in the Preferences folder inside the System folder The benefits to using these functions are as follows Simply use Calibration File Send to reenter the CCD pixel position value rather than manually locating and reentering the value Once Calibration File Send is executed the instrument serial number is added to all sample files and is visible when sample files are opened in the annotative view This is useful for tracking and troubleshooting particularly when more than one instrument is in operation See
111. 0W C CHILLER GS Run 60W D CHILLER GS Run 2140V A CHILLER GS Run 2140V D CHILLER GS Run 2140V C CHILLER 1 Use virtual filter set E modules with ABI PRism dRhodamine Terminator Cycle Sequencing Kits or the dRhodamine Matrix Standards Kit only Use virtual filter set D and F modules with GeneScan and NED dyes 2 These chiller modules are recommended for PCR Single stranded Conformation Polymorphism Analysis SSCP 9 48 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com DyeSet Primer Files What are DyeSet Primer Files DyeSet Primer File Naming Conventions For sequencing applications only dye set primer files Must be specified on sample sheets for data to be analyzed automatically Contain the information used to Compensate for differences shifts in sample mobility Interpret what each dye color in a set represents Each of the dyes used in a set affects the electrophoretic mobility of cycle sequencing extension products differently The relative mobility of the dye labeled fragments is specific to each sequencing chemistry Under the same set of run conditions the mobilities are very reproducible ABI PRISM DNA Sequencing Analysis Software compensates for these mobility differences by applying the mobility shift information contained in dye set primer files As a result evenly spaced peaks are presented
112. 2 2 gel sapcers See spacers gel temperature determining 3 36 9 43 modifying 9 45 viewing in Status window 3 36 Gel window description of 3 42 9 55 GelDoc II utility 1 19 gels factors that affect gel quality A 4 to A 8 age of the gel A 8 air bubbles A 8 rate of polymerization A 7 reagent purity A 5 to A 6 preparing A 2 removing from glass plates 3 45 selecting a gel formulation 2 2 troubleshooting gel and gel image problems 4 3 to 4 4 using high quality gels importance of 2 3 using new glass plates 2 5 See Also gel recipes GenBase software 1 20 GeneAssist Sequence Analysis System 1 21 general settings description of and how to change 5 15 to 5 16 GeneScan analysis parameters folder location and description 3 52 GeneScan Analysis Software automatic data analysis 9 38 description of software 1 18 fragment kit and reagent part numbers C 8 to C 11 matrix files See matrix files 6 1 optimizing electrophoresis conditions 4 34 to 4 35 run sheets about 9 29 to 9 30 preparing 9 31 to 9 37 setting preferences 5 13 to 5 14 sample sheets about 9 20 to 9 21 entering information 9 26 importing exporting information 9 27 preparing 9 22 to 9 25 setting preferences 5 12 GeneScan size standard folder location and description 3 52 GenoPedigree software 1 20 Genotyper software 1 20 getting started if unfamiliar with instrument new user information 1 3 safety information 1 4 glass plates avoid organic solvent 2 7 before usi
113. 32 36 and 48 well shark s tooth and 24 32 and 36 well square tooth Data is collected from 194 channels during each scan ABI PRISM 377 18 DNA Sequencer The ABI PRISM 377 18 DNA Sequencer is the lowest cost lowest throughput version of the instrument It can run up to 18 lanes on a single gel ABI PRISM 377 DNA Sequencer with XL Upgrade The ABI PRISM 377 DNA Sequencer with XL Upgrade increases the number of samples that can be analyzed simultaneously Increased throughput is made possible by modifying the instrument to collect data from 388 channels instead of 194 during each scan About This Instrument 1 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 1 12 About This Instrument The XL Upgrade can accommodate the following combs 48 and 64 well shark s tooth combs for sequencing applications 48 50 64 and 66 well for GeneScan applications You can still use 36 well or other lower lane density combs if desired Refer to the ABI PRISM 377 DNA Sequencer XL Upgrade User s Manual P N 904412 for more information ABI PRISM 377 DNA Sequencer with 96 Lane Upgrade The ABI PRISM 377 DNA Sequencer with 96 lane Upgrade increases the number of samples that can be run on each gel Increased throughput is made possible by re engineering the instrument to collect data from 480 channels instead of 388 for the ABI PRISM 377 DNA Sequencer with XL Upgrade or 194 for the AB
114. 32 36 lanes XL Scan 48 50 64 66 lanes XL Scan 48 64 lanes 96 Lane Scan 96 lanes 96 Lane Scan 96 lanes Note If this is an XL or 96 lane upgrade instrument the appropriate value for the Run Mode field is selected automatically when this parameter is set The run sheet for the standard ABI PRISM 377 instrument does not have a Run Mode field Selecting the IMPORTANT Select the number of lanes before selecting the sample sheet If the default Sample Sheet umber of lanes is less than the number of samples the sample information on the run sheet will be truncated For example if you entered 36 samples on the sample sheet but the default number of lanes on the run sheet is 24 only the information for the first 24 samples will be imported to the run sheet if the sample sheet is selected before the number of lanes is changed to 36 To select a sample sheet Step Action 1 Open the sample sheet pop up menu and select the sample sheet prepared for this run Once selected the information on the sample sheet is imported into the run sheet IMPORTANT If the pop up menu lists lt none gt only software cannot find the folder that contains the sample sheets To correct this see Setting Folder Location Preferences on page 9 5 The information imported from the sample sheet cannot be changed on the run sheet If changes are made to the sample sheet after it has been selected on the run sheet you must reimport
115. 388 for ABI PRISM 377 XL instruments numbered 0 to 387 480 for ABI PRISM 377 96 lane instrument numbered 0 to 479 The type of comb used determines the number of channels a lane will include For example one lane of a 36 well comb is equivalent to approximately 5 channels To perform a plate check you open a new run sheet select a plate check module and click the Plate Check button on the run sheet A Scan window opens automatically and you observe the window for approximately 2 minutes Scan Window C Freeze Updates Current Scan Number 16 Relatively flat scan lines as shown in the figure above indicate the plates are clean and there are no contaminants in the read region of the gel If peaks appear either the plates are dirty or the gel is contaminated The plates can be cleaned and the plate check repeated If peaks still appear the gel is probably contaminated About This Instrument 1 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The PreRun 1 16 About This Instrument If the gel is contaminated you can Load anew gel Determine which lanes are safe to load with samples and which lanes should be left empty This is referred to as skipping lanes Terminate the plate check fill the buffer chambers start the prerun and allow the gel to electrophorese for a few minutes while watching the Sc
116. 5 recommended dishwashers 3 8 I injection device fixture for gel preparation 3 14 Artisan Technology Group Quality Instrumentation O organic solvents and glass plates 3 7 P plates cleaning cleaning by hand 3 6 to 3 7 recommended dishwashers 3 8 using adishwasher 3 6 gasket mark 3 5 identifying front and back 3 5 using new glass plates 3 5 pouring gels See gel pouring methods S solvents organic and glass plates 3 7 spacers cleaning 3 8 placement of 3 11 U unmounted plates gel pouring method 3 4 Guaranteed 888 88 SOURCE www artisantg com Index 1 rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at ww
117. 6 cm Lanes 24 v PreRun Module Seq PR 36A 1200 Y Run Module Seq Run 36A 1200 v DE Antona ee With Analysis software E Auto Print E not selected Conce C If the analysis software is Then selected click OK to return to the run sheet not selected a Open the Autoanalyze with pop up menu and select Other b Inthe dialog box locate the ABI PRISM DNA Sequencing Analysis Software and click Open c Close the current run sheet and create a new run sheet Note Changing the preference has no affect on run sheets created prior to the change On the run sheet verify that the Auto Analyze boxes are selected for each non matrix standard sample If the boxes are not selected a Reopen the sample sheet by clicking the icon next to the sample sheet pop up menu Sample Sheet x Click this icon b Select a dye set primer file for each sample that you want analyzed automatically c Close and save the sample sheet Open the sample sheet pop up menu and select lt none gt Open the sample sheet pop up menu and reselect the sample sheet to update the information If matrix standard samples are being run deselect Auto Analyze for all matrix standard samples Select Auto Print for analyzed data to be printed automatically Proceed to Starting the Run or Closing the Run Sheet on page 9 37 Data Collection Software 9 35 Arti
118. 610 A 22 Gel Recipes Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Storing Reagents and Stock Solutions Acrylamide Good quality acrylamide can be stored dry at room temperature for up to one year 40 stock solution of acrylamide and bisacrylamide 19 1 or 29 1 can be stored at 4 C for up to one month Use distilled deionized water only Using the premixed powders saves time and minimizes your exposure to the solid form of the neurotoxins acrylamide and bisacrylamide Bisacrylamide Store bisacrylamide dry at room temperature for up to one year 40 stock solution of acrylamide and bisacrylamide 19 1 or 29 1 at 4 C for up to one month WARNING CHEMICAL HAZARD Acrylamide and bisacrylamide are neurotoxins Avoid inhalation and skin contact Wear gloves at all times and work in a fume hood when handling acrylamide solutions and use appropriate precautions to avoid inhalation of crystalline acrylamide TEMED Because it is hygroscopic this initiator gradually accumulates water which increases the rate of oxidation Water free TEMED gt 99 pure stored in a tightly sealed container at room temperature should be good for up to six months WARNING CHEMICAL AND FIRE HAZARD TEMED is extremely flammable and can be very destructive to the skin eyes nose and respiratory system Keep it in a tightly closed container Avoid inhalation and c
119. 7 6 FAM Phosphoramidite 85 mg 401533 TET Phosphoramidite 100 mg 401526 HEX Phosphoramidite 105 mg Fluorescent For post synthesis labeling of primers containing a 5 Aminolink 2 NHS Esters 400981 TAMRA NHS Ester 5 mg 60 uL in DMSO 400980 ROX NHS Ester 5 mg 60 uL in DMSO 400808 Aminolink 2 0 25 g Matrix Standard Sets 401114 Dye Primer Matrix Standards Kit Filter Set A for NHS ester labeling Contains one tube each of 5 FAM JOE TAMRA and ROX labeled DNA 402792 F JdNTP Matrix Standards Contains one tube each of R110 R6G TAMRA and ROX labeled DNA 401546 Fluorescent Amidite Matrix Standards Kit Filter Set C for fluorescent phosphoramidite labeling Contains one tube each of 6 FAM TET HEX TAMRA and ROX labeled DNA 402996 NED Matrix Standard Used in combination with the 5 FAM JOE and ROX dyes in the Dye Primer Matrix Standards Kit or the 6 FAM HEX and ROX dyes in the Fluorescent Amidite Matrix Standards Kit Parts and Accessories C 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Fluorescent Genotyping A and B Contains six fluorescent labeled PCR primer pairs labeled with HEX TET amp FAM two control DNAs CEPH 1347 02 and 1347 10 and a ready made mix of PCR reagents containing AmpliTaq Gold DNA Polymerase GeneAmp PCR Buffer Il dNTPs and magnesium chloride Also in
120. 7 3 Summary of Chemistries so vit rer saune nese sade pA wee eee dee ed Meee een ee 7 4 Color Guide for Data Display Windows 00 cece eee eee eens 7 4 Making an Instrument File for Virtual Filter Set A from Matrix Standards 7 6 Making an Instrument File for Virtual Filter Set E from Matrix Standards 7 10 Checking Instrument Files 2 0 0 cece cette tenes 7 16 Making an Instrument File from a Sample File 0 0 0 0 e eee eee eee 7 18 Storing and Backing Up the Instrument File 0 0 0 eee eee eee 7 19 v Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Adding or Replacing a Matrix in an Existing Instrument File 7 20 Correcting Errors in Matrix Creation 0 0 cece cc rererere 7 22 8 System Maintenance ccc cece eee eee e cence es On Chapter Contents s sai b cis aie neti aiai ioi ae aad Ghia ea Sp vend Ware he aed Rada wee 8 1 Instrument Maintenance Recommendations 0 0 eee eee ee eee 8 2 Cleaning Instrument Accessories 0 0 0 eee ce eee e ene n ees 8 3 Refilling the Water Reservoir 2 0 cee eee nee tenet nee ees 8 5 Replacing the Upper Buffer Chamber Gasket 0 0 00 cee cece eee eee eee 8 6 Replacing Electrophoresis Cables and Electrodes 00 0 0c cece ee eee eee 8 12 CCD Pixel Position Value oses s4 84s5 G5 Gee cd eee eee EN Sew eRe
121. 77 DNA Sequencers P N 4303188 The signal from the large peak is off scale because of sample overloading In the example shown in Figure 6 1 the peak showing bleedthrough is actually off scale Figure 6 2 2000 Figure 6 2 Raw data from the bleedthrough example shown in Figure 6 1 Keep peak heights between approximately 150 and 4000 RFU If sample data is off scale do one of the following Rerun the samples loading a smaller volume Dilute the samples and rerun them Making Matrix Files for GeneScan 6 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Elevated Interpeak Figure 6 3 shows a typical example of an elevated interpeak baseline In this example Baseline the green baseline is elevated in the region between two large black peaks representing the yellow dye signal because too much green signal is subtracted from the yellow signal see Figure 6 4 on page 6 5 The GeneScan software uses these low data points to calculate the baseline for the green signal Therefore the original baseline is elevated Note Ifthe baseline is sufficiently elevated random fluctuations in the baseline can lie above the Peak Amplitude Threshold and might be falsely interpreted as product peaks If you suspect that an elevated interpeak baseline is caused by matrix problems inspect the data before baselining This can be done by reanaly
122. 8 26 you will now be asked to specify various folder locations See Folder Location Preferences in Chapter 5 Setting Preferences System Maintenance 8 27 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Adding Non Applied Biosystems Software Recommendations INIT Troubleshooting Procedure 8 28 System Maintenance IMPORTANT We strongly recommend that you do not install other programs on the Macintosh computer that runs the instrument Some programs can interfere with instrument operation If additional software is installed keep SAM activated to check for viruses described on page 8 21 Additional recommendations Do not install games or screen savers onto the hard disk Do not install programs that require additional control panels CDEVs or extensions INITs This includes games with custom sound or graphics Unnecessary INITs can be turned off using the Extensions Manager INITs Extensions and CDEVs reside in the extensions and control panel folders in the System folder and alter the programming code of the system Compatibility is guaranteed only for the INITs shipped with the ABI PRISM 377 DNA Sequencer Even seemingly innocuous INITs like SuperClock and After Dark can interfere with system operation If you suspect there is a problem with a specific INIT follow the troubleshooting procedure listed below If you suspect there is a
123. 9 1 Polyacrylamide Gels Protocol and Run Conditions A 11 PAGE PLUS Gels Protocol and Run Conditions A 13 Long Ranger Gel Solution Protocols A 15 MDE Gel Protocol for SSCP A 18 Polyacrylamide Gel Solution Protocols A 19 Supplier Information A 22 Storing Reagents and Stock Solutions A 23 Gel Recipes A 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com List of Gel Recipes and Recommendations List of Recipes The following gel recipes for sequencing applications are provided in this appendix Gel Formulation See page Polyacrylamide Generic 19 1 A 19 29 1 4 5 29 1 4 25 A 11 for SSCP Long Ranger 5 0 4 75 Long Ranger Singel A 15 for SSCP PAGE PLUS 4 8 5 25 ee Mutation Detection gel MDE for SSCP A 18 SSCP Single strand Conformation Polymorphism Recommendations Gel formulations that provide longer read lengths when compared to read lengths for Longest from 19 1 polyacrylamide gels of the same plate size and run speed are listed in the Read Length following table Plate Size and Expected Read Run Speed Recommended Gel Formulations Length Ranges 36 cm well to read 4 5 29 1 polyacrylamide WTR plates 4 8 PAGE PLUS 650 800 1200 scans hr 5 0 Long Ranger concentrate or Singel gel forms 36 cm WTR plates 4 5 29 1 polyacrylamide 550 700 2400 scans hr 48 cm
124. AL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Wear appropriate protective eyewear clothing and gloves Obtain a copy of the MSDS from the manufacturer Prepare this solution before you start or as needed based on the procedures in this chapter Prepare loading solution fresh daily by mixing the following ingredients in a 5 1 ratio formamide EDTA with blue dextran The amount you will need to prepare will vary depending on the number of lanes used Instrument Operation 3 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Cleaning the Gel Plates Before Loading the Gel How to Carry the IMPORTANT When carrying a cassette with plates always hold the cassette by the sides Cassette with Plates with both hands Do not carry the cassette by the top bar The weight of the plates may cause the bar to break resulting in broken glass plates Do not hold cassette here to carry glass plates Hold cassette by the sides with both hands to carry glass plates P Cleaning the Plates Follow this procedure to clean the outside surface of the glass plates before loading Before Using the Gel the gel on the instrument IMPORTANT Wear gloves throughout this procedure for your protection and
125. Ac 21M13 DyeSet Primer pop up menu DyeSet Primer lt none gt Instrument File lt none gt v DP6 Ac M1 3Rev DP6 Ac SK DP6 Ac SP6 DP6 Ac T3 DP6 Ac T DT4 Ac A Set AnyPrimer DT 4 Ac B Set AnyPrimer DT6 Ac A Set AnyPrimer DT6 Ac B Set AnyPrimer If Then you are finished click OK to save your changes or Cancel to quit without making any changes you wish to change more preferences open the Page pop up menu and select another Preference 5 10 Setting Preferences Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Sequencing Run Sheet Preferences Setting Sequencing The following parameters can be set as preferences for sequencing run files Run Sheet 4 Operator Preferences Well to read WTR distance in centimeters Lanes PreRun module Run module Autoanalyze Auto Print gt gt gt gt To change sequencing run file preferences Step Action 1 If the Preferences dialog box Then is not open open the Windows pull down menu select Preferences and then select Sequence Run Defaults is open open the Page pop up menu and select Sequence Run Defaults 2 To enter an operator s name type the name directly into the Operator field Preferences Page Sequence Run Defaults kal Operator Jody Carrillo WTR cm
126. Analyze The gel file is closed sent to the analysis software and then analyzed Data collection software automatically quits and the run is terminated Note To use the Stop amp Analyze feature the run sheet must be configured for automatic data analysis before the run is started The run cannot be resumed after selecting Stop amp Analyze 3 40 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The amount of time it takes for peaks to appear varies and is dependent upon the well to read distance type of gel and run speed General guidelines are listed below Approximate Amount of Time Well to Read Run Speed Before Peaks are Detected Distance cm scans hour Sequencing GeneScan 12 1200 2400 N A 10 20 minutes 36 1200 40 minutes 20 25 minutes 36 2400 20 minutes 20 25 minutes 48 1200 1 hour N A Monitoring the Run Four types of windows are available for viewing data and instrument status Data windows are Scan Gel Instrument windows are Status Electrophoresis History Refer to Chapter 9 Data Collection Software for more information on these windows Note To use as little RAM as possible we recommend leaving a minimum number of windows open for example only the Status and Scan windows The Gel window requires the largest amount of RAM to display Scan Window The Scan win
127. Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com About Automatic Data Analysis Overview For Sequencing Applications For GeneScan Applications Data can be analyzed and printed automatically or manually after a run When the software is set up for automatic data analysis the data collected during the run and all other information required for analysis is transferred to the analysis software program automatically at the end of the run The analysis software then automatically analyzes the data and prints the results The information that must be specified for automatic data analysis is application specific Requirements are listed below Auto analyze must be selected to use the Stop amp Analyze feature during the run Described on page 9 39 To perform automatic data analysis after a sequencing run the following information must be selected on sequencing sample and run sheets On the sequencing sample sheet A dye set primer file Selecting a dye set primer file on the sample sheet automatically places an X in the corresponding Auto Analyze box on the run sheet An instrument file Onthe sequencing run sheet Auto print optional In addition the analysis program ABI PRISM DNA Sequencing Analysis Software must be selected on the Sequence Run Defaults Preference This tells the data collection software where to send the data when the run is finis
128. Calibration File Send 4 27 to 4 28 CCD Gain function description of 3 49 CCD offset function description of 3 50 CCD pixel position value 3 49 4 25 location of 4 26 reentering 4 26 using Calibration File Make 4 25 using Calibration File Send 4 25 channels number of in each lane 1 15 chemistries chemical abbreviations used in gel recipes A 9 gels used for GeneScan runs 2 2 gels used for sequencing runs 2 2 7 4 sequencing gel recipes A 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com chiller modules description of 9 44 installing 9 44 to 9 45 chiller modules folder location and description of 3 51 clamps part numbers C 2 cleaning instrument accessories 8 3 to 8 4 cleanup afterarun 3 45 cold boot instrument function 3 50 cold boot procedure 4 23 collection time modifying 9 45 suggested times 3 31 combs cleaning 2 8 inserting 96 lane comb 3 6 inserting shark s tooth 3 6 inserting square tooth 3 6 part numbers C 3 computer See Macintosh computer contaminants of acrylamide A 5 buffers A 6 Tris A 6 urea A 6 coolant system clogs removing 4 32 to 4 33 customer support e mail address 1 6 internet address 1 9 telephone fax 1 6 to 1 9 1 6 to 1 10 D data deleting data files 8 18 to 8 19 viewing 9 53 to 9 57 Gel window 3 41 9 55 Scan window 3 41 9 55 data analysis about automatic analysis 9 38 designating Settings folder for auto analysis 3 52
129. Common Problems The two most common problems that can occur during matrix creation are Signal too weak Incorrect files or chemistry selected resulting in an error message stating the matrix was not made successfully Signal Too Weak If the signal size for any of the data is too small an error message appears and the matrix is not made To correct for weak signal Step Action 1 Open the Sequencing Analysis program Open sample file for the standard in the Sample window Open the Window menu and select Raw Data 2 3 4 Find a data range with about 1500 points with reasonable signal strength Write down the start and end points for that range If the file does not contain enough good data run a new set of matrix standards Repeat the Make Matrix process or the Add Replace Matrix process on page 7 20 using the new start point and data range numbers Incorrect Files or _ If any of the files selected are obviously incorrect or if the wrong chemistry button was Chemistry selected an error message appears and the matrix is not made To correct file or chemistry selection Step Action 1 Repeat the Make Matrix process selecting the correct chemistry button for the correct set of matrix sample files Use the gel file to verify that matrix sample files contain the dye that the file indicates Color Guide for Data Display Windows on page 7 4 explains the co
130. HD ABI Prism 377 Firmware Image LA settings Folder Macintosh HD System Folder ABI Folder C GeneScan Analysis Parameters Macintosh HD GeneScan 65 Parameters Folder C GeneScan Size Standard Folder Macintosh HD GeneScan 0 65 Standards Folder Co Click in these boxes to select and change a folder location 3 52 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Step Action 3 Using the pop up menu in the dialog box that is displayed locate and select the folder the appropriate folder IMPORTANT To automatically analyze data with ABI PRism DNA Sequencing Analysis Software on the same computer used for data collection the Settings Folder must be designated as the ABI Folder shown below The text above the menu indicates the folder location preference currently being set and corresponds to the select button at the bottom of the box Use this pop up Select Settings Folder menu to locate the desired folder Si System Folder Y Macintosh fm ABI Folder Highlight the new 7 Acrobat Search Resources folder by clicking CAECSoft it once Co Apple Menu Items 7 AppleShare Folder Claris Control Panels ee Control Panels Disabled Click here to select the new folder do Select ABI Folder not click Open 4 Click the Select box underneath
131. I PRISM 377 DNA Sequencer In addition the scan region has been increased from 6 inches to 7 25 inches The 96 lane upgrade includes a 100 well shark s tooth comb and a new front glass plate The new front glass plate has a bevel where samples are loaded The bevel increases the thickness of the gel from 0 2 mm to 0 4 mm in this region Lower lane density combs can still be used but only with the original front glass plates provided with the standard ABI PRISM 377 instrument Refer to the ABI PRISM 377 DNA Sequencer 96 Lane Upgrade User s Manual P N 4305423 for more information Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Theory of Operation How the Instrument The ABI PRISM 377 DNA Sequencer is an automated instrument designed for Works analyzing fluorescently labeled DNA fragments by gel electrophoresis Gel electrophoresis is the movement of charged molecules through a gel solution Such as polyacrylamide in an electrical field It is used to separate DNA fragments by size The instrument functions the same way for both sequencing and GeneScan applications The main differences between these applications is in the preparatory chemistries used and the type of analysis performed on the resulting data To use the instrument DNA fragments labeled with up to four different fluorescent dyes are combined and loaded into one lane on a vertical slab gel made of polymerized a
132. ISM 377 folder We suggest renaming the folder ABI PRISM 377 lt version number gt For example ABI PRISM 377 v2 1 Open the Preferences folder located inside the System folder Select the file named ABI 377 ABI 377XL ABI 377 96 or ABI 377 18 as appropriate and rename it We suggest adding the suffix v lt version number gt For example Preferences v2 1 Proceed to Downloading Data Collection Software from Our Website Downloading from To download the software from our website the Website Step Action 1 Access the ABI PRISM 377 DNA Sequencer website at www appliedbiosystems com techsupport Click Download Software Click the 377 folder icon If not already on the hard drive click on the DiskCopy lt version number gt hqx file This utility program is required to create a floppy disk Click the appropriate data collection software hqx file For example ABI PRISM 377 v2 1 image hqx 8 26 System Maintenance Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Step Action 6 To copy the software onto a floppy disk a Double click the DiskCopy icon b Open the Utilities menu and select Make A Floppy c Select the data collection software image from the menu and click Open d When prompted insert a blank floppy disk into the hard drive e When finished open the F
133. Includes Detailed instructions for two methods of pouring gels Recommendations and instructions for preparing a variety of gels that can be used on this instrument Storage recommendations for reagents and stock solutions Factors that affect gel quality and read lengths for sequencing Supplier information Chapter 3 Using the Instrument Provides detailed instructions for setting up and operating the instrument for both GeneScan analysis software and sequencing analysis software applications Note Chapters 2 and 3 and Appendix A are bound separately for your convenience They can be removed from the binder and used separately as quick reference guides for pouring gels and using the instrument About This Instrument 1 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Safety Information Site Preparation and Safety Guide Safety Warnings in this Manual Ergonomic Hazard 1 4 About This Instrument The ABI PRISM 377 Site Preparation and Safety Guide P N 903393 was sent to you prior to instrument installation This guide includes important safety information that should be read by all users before they operate the instrument Topics covered in the guide include Anexplanation of the safety labels affixed to the instrument Laser safety including hazards and safe operation requirements Chemical safety including a waste profile and
134. Instrument Files Step Action 1 Use the Finder to make sure the new instrument file is stored in the ABI Folder inside the System Folder If you saved the file to a different location drag it to the ABI folder now To be used by the Sequencing Analysis program the instrument file must be in the ABI folder Clean up the ABI folder by deleting any invalid instrument files Put a backup copy of the instrument file on a server or a disk and put the disk in a safe location It is a good idea to put the raw sample files for the standards in the same place Making Instrument Files for Sequencing 7 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Adding or Replacing a Matrix in an Existing Instrument File Introduction Use the procedure described below to Adda matrix to an incomplete instrument file Replace an existing matrix Make an additional instrument file for testing purposes Note Be sure to make a backup copy of the original instrument file before you modify it Adding or Replacing a Matrix oo sete a To add or replace a matrix in an existing instrument file Step Action 1 Open the DataUtility program The program icon looks like this The program is located in the Utilities folder inside the Sequencing Analysis folder 2 Choose Make Matrix from the Utilities menu The Make Matrix dialog box appears Make M
135. Mounted Plates and the Gel Injection Device 2 18 Pouring Gels Prepare the gel solution now See Selecting a Gel Formulation on page 2 2 for guidelines Gel recipes are listed in Appendix A Gel Recipes IMPORTANT The polymerizing reagents ammonium persulfate and TEMED are the catalysts that initiate polymerization Once these reagents are added to the gel solution you must work quickly to pour the gel Polymerization can occur within 15 minutes At this point in the gel preparation procedure the glass plates spacers and comb should be mounted in the gel cassette The gel injection device should be attached to the plates The entire assembly should be resting on a level raised support WARNING CHEMICAL HAZARD Long Ranger gel solution contains acrylamide Acrylamide is a neurotoxin Avoid skin contact with Long Ranger gel solution because acrylamide can be absorbed through the skin Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves WARNING CHEMICAL HAZARD Acrylamide and bisacrylamide are poisons neurotoxins irritants carcinogens and possible teratogens Acrylamide and bisacrylamide sublime the solids release toxic vapor and are harmful if swallowed inhaled or absorbed through the skin Effects are cumulative When handling always wear protective equipment lab coat safety glasses and chemical resistant gloves and us
136. NPT 1 4 in male barbed fittings NESLAB P N 126000000007 Any external water bath that meets these requirements should work properly The NESLAB Model RTE111 meets these requirements and has been tested with the instrument Refer to the user manual for the water bath you select follow proper procedures and ensure that all product safety and regulatory requirements applicable to your location are met Thermistors To perform SSCP at temperatures less than 22 C the instrument must have 30 k ohm thermistors installed Some instrument may have 100 k ohm thermistors which must be replaced to use an external water bath To determine which thermistors are installed on the instrument check a Log file from a recent run using ABI PRISM Data Collection Software version 2 1 or higher The configuration information in the Log lists the type of thermistors installed If necessary schedule a service call with your Applied Biosystems service engineer to replace the 100 k thermistors with 30 k thermistors Software ABI Prism Data Collection Software version 2 1 and above contain the features Requirements required to accommodate the external water bath including chiller modules Performing runs at subambient temperatures with an external water bath requires the specification of chiller modules on the run sheet Standard modules are located in a folder called Modules At system installation the software is typically set up so that the
137. Name Project 1 Second Sample Name Second Sample Name Second Sample Name 6 F AM Matrix Standard 6 FAM Matrix Standard TET Matrix Standard TET Matrix Standard The diamond indicates the color of the size standard Checkboxes indicate the dyes run in each lane Figure 3 5 Example of a completed GeneScan sample sheet Sample Sheet Save and Close the To save and close the sample sheet Step Action 1 Do one of the following a Open the File menu and select Close b Click the box in the upper left hand corner of the window and then click Save A dialog box showing the default sample sheet file name and the location where the sample sheet will be stored is displayed a Change the file name now if desired b Click Save IMPORTANT Although it is an option we do not recommend changing the storage location of the sample sheet If the location is changed software will not be able to locate the sample sheet when you set up the run sheet Folder where sample sheets are stored fu Sample Sheet 5 fu Sample Sheet fg Sample Sheet 5 fa Sample Sheet 5 15 98 3 18 P N n Sample Sheet 5 15 98 3 Desktop jew Cancel Save this document as Sample sheet file name
138. P Set R110 R6G and TAMRA 3 3 and 12 nmol 3 x 30 pL 401896 R110 dUTP 6 nmol 2 x 30 pL 401897 R6G dUTP 6 nmol 2 x 30 pL 401895 TAMRA dUTP 24 nmol 2 x 30 pL 402793 F JdCTP Set R110 R6G and TAMRA 3 3 and 12 nmol 3 x 30 pL 402795 R110 dCTP 6 nmol 2 x 30 pL 402796 R6G dCTP 6 nmol 2 x 30 pL 402794 TAMRA dUTP 24 nmol 2 x 30 pL Note TAMRAJdNTP is supplied at a concentration four times higher than R110 dNTP and R6G dNTP because it produces approximately four times less signal Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Fluorescent dNTP Each kit listed below includes a GeneAmp kit as specified 100 reactions along with PCR Kits an FJdNTP set that contains 30 uL each of R110 dNTP 3 nmol R6GJdNTP 3 nmol and TAMRA dNTP 12 nmol N808 0220 GeneAmp PCR Reagent Kit with AmpliTaq DNA Polymerase with F JGUTP Set N808 0221 GeneAmp PCR Core Reagents with F JdUTP Set N808 0222 GeneAmp Thermostable r7th Reverse Transcriptase RNA PCR Kit with F JdUTP Set N808 0223 GeneAmp PCR Reagent Kit with AmpliTaq DNA Polymerase with F JdCTP Set N808 0224 GeneAmp PCR Core Reagents with F JdCTP Set N808 0225 GeneAmp Thermostable r7th Reverse Transcriptase RNA PCR Kit with F JdCTP Set Fluorescent For direct 5 end labeling on an automated DNA synthesizer Phosphoramidites 40152
139. Printing Files si cesa eee aes teat awa A ole aaa a de en oe ers 9 58 Quitting the Data Collection Program 0 0 cece tenes 9 59 Data Analysis OVerview ecse 26644 ectige aes eee eed eee Ode ee eee 9 60 Gel R cipls s oie veer ieee ete towe wads eina a e A Appendix Contents 4 055 sernpri wake ha RE ne ae ee E AEREE E A A 1 List of Gel Recipes and Recommendations 0 0 cece eee eee eee A 2 Sequencing Chemistries Released in 1997 00 0 cee eee A 3 Factors that Affect Gel Quality and Read Lengths 0 0 0 cee eee eee eee A 4 Chemical Abbreviations Used 0 0 0 0 cee ect eee eens A 9 Preparing TBE Butter icci css cence ond op eee beeen esa eee na ehee eke tan baata A 9 Deionizing Formamide 0 cece ae eee sag a ewes a eee ane hha ia A 10 29 1 Polyacrylamide Gels Protocol and Run Conditions 00 5 A 11 PAGE PLUS Gels Protocol and Run Conditions 0 0 00 00 eee eee eee A 13 Long Ranger Gel Solution Protocols 0 0 0 c cece ee renner A 15 MDE Gel Protocol for SSCP 2 cece eee een eee A 18 Polyacrylamide Gel Solution Protocols 0 c cece eee ee een eee A 19 Supplier Information s s srs isss sn cee cent e eens A 22 Storing Reagents and Stock Solutions 2 0 0 0 eee cece eee eee A 23 B Subambient Temperature Operation 000000044 Bel Appendix Contents 3 s 20 sc bees one ood od Sa SAAS ea Sue ede ee B 1 Purpose of an Extern
140. Project Information For BioLIMS software users only Project information 5 20 required to properly identify data transferred to BioLIMS Where are Preferences Stored Location Preferences are stored in a file that has one of the following names ABI 377 for standard ABI PRISM 377 instruments ABI 377XL for ABI PRISM 377 instruments with the XL upgrade ABI 377 96 ABI PRISM 377 instruments with the 96 lane upgrade ABI 377 18 ABI PRISM 377 18 instruments The preferences file is located inside the Preferences folder which is located inside the System Folder on the hard disk 5 2 Setting Preferences Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Viewing and Modifying Preferences How To The information in the preferences file cannot be viewed or modified by double clicking on the file icon To view and or modify preferences Step Action 1 Launch the data collection software 2 Open the Window menu and select Preferences to open the preferences menu Window Status Log Sample Sheet Run Scan Gel Image Electrophoresis History Manual Control Preferences A Folder Locations Default File Names Sequence Sample Sheet Defaults Sequence Run Defaults GeneScan Sample Sheet Defaults GeneScan Run Defaults General Settings Dye Indicators Project Info 3 Select a preference A dial
141. Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Reclean the Plates Peaks in the scan lines during a Plate Check indicate contaminants on the glass in the read region or contaminating fluorescence in the gel Clean the plates again as described below and repeat the plate check To clean the plates and repeat the plate check Step Action 1 Click Pause to pause the plate check 2 Open the right front panel of the instrument and remove the gel cassette WARNING LASER HAZARD Exposure to direct or reflected laser light at 40 mW for 0 1 seconds can burn the retina and leave permanent blind spots Never look directly into the laser beam or allow a reflection of the beam to enter your eyes Carefully clean both sides of the glass with deionized water and Kimwipes particularly the read region Remount the cassette in the electrophoresis chamber Close the front panel of the instrument Click Resume to check the plates again NI OJO A If Then the plates are clean a Click Cancel and terminate the plate check b Leave the run window open c Proceed to Installing the Upper Buffer Chamber on page 3 15 peaks are still present the gel is probably contaminated Choose one of the following Determine which lanes are contaminated and leave those lanes empty when loading samples See Skipping Lanes on page 3 14 to determine which
142. R110 dR6G dTAMRA dROX AO gt O For each matrix standard sample start with the default value of 2000 for the start point Start with the default value of 1500 for the number of data points to analyze Note If the default values do not work follow the instructions for using other values in steps 8 and 9 below Making Instrument Files for Sequencing 7 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To make the Dye Primer Matrix continued Step Action 5 Click New File A dialog window appears as shown below Name the file dRnod_BigDye or another appropriate name and save it in the ABI folder within the System folder ABI Folder Y Shadow ee Generic Matrix 2 Eject te Koshka s Matrix Seq Analysis Command File Desktop Seq Analysis Error File Shadow s Matrix Name for new matrix file Cancel dRhod_BigDye 6 The Make Matrix dialog box should look like that shown below Make Matrix 001 E 21 dR110 matrix std Start at 17 dR6G matrix std Start at G 19 dTAMRA matrix std Start at 23 dROH matrix std Start at Points dRhod_BigDye Instrument Comment Dye Primer Matrix Taq Terminator Matrix OT Terminator Matrix a Type information in the Instrument and Comment text boxes e g the instrument serial number or name and the date the matrix was created
143. RCE www artisantg com Observation Possible Causes Recommended Actions Faint or no signal from sample DNA and from positive control continued from previous page PCR Master Mix not well mixed before aliquoting Vortex PCR Master Mix thoroughly Primer concentration too low Use the recommended primer concentration Primers degraded Use new primers Note Preincubation at 95 C for 5 10 minutes should inactivate proteases or nucleases Note To prevent primer degradation during storage store primers at 15 to 25 C either lyophilized or in TE Avoid excessive more than 3 4 freeze thaw cycles Too little free Mg in reaction Check that you added sufficient total Mg given concentration of the dNTPs and EDTA Note Free Mg Total Mg Total dNTP 2 EDTA Incorrect pH Verify buffer pH and buffer concentration Wrong PCR tube Use GeneAmp Thin Walled Reaction Tubes for DNA Thermal Cycler 480 MicroAmp Reaction Tubes with Caps for the GeneAmp PCR Systems 9600 and 2400 MicroAmp Base used with tray retainer set and tubes in GeneAmp PCR System 9600 or 2400 Remove MicroAmp Base from tray retainer set and repeat amplification Verify GeneAmp PCR System protocols and programmed parameters Refer to the thermal cycler user s manual and check instrument calibration Tubes not seated tightly in the thermal cycler d
144. Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com General Sample The following general information will help you prepare sample sheets Sheet Information Sample information is used by the software to identify each individual sample when Sample Files described on page 9 60 are generated after a run Therefore it is critical that each sample name be entered in the field whose number corresponds to the gel lane in which the sample is loaded If the same type of run is performed repeatedly on the same instrument you can reduce the time spent setting up GeneScan sample sheets by setting GeneScan sample sheet default preferences See Save Time by Setting Sample Sheet Preferences on page 9 22 and Chapter 5 for more information Information can be imported from tab delimited text files for instance files generated by a database The sample sheet can be exported as a tab delimited text file to database spreadsheet or word processing programs Refer to Importing and Exporting Sample Sheet Information on page 9 27 for more information The same sample sheet can be used for more than one run if the information required is the same An existing sample sheet can also be used as a template to create new sample sheets by opening the file saving it under a different name Save As command under the File menu and then modifying the new file Sample sheets can be p
145. a 0 2 um cellulose nitrate filter to filter the solution o Degas the filtrate by one of the following methods Purging with argon or helium Applying a vacuum for at least five minutes IMPORTANT If the plates are not clean and mounted in the gel cassette or other device clean and mount them now before adding the polymerizing agents to the gel solution Instructions are listed in Chapter 2 Pouring Gels A 16 Gel Recipes Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Adding the polymerizing reagents Step Action 1 Add fresh 10 APS to the gel solution Swirl gently to mix Be careful to avoid introducing air bubbles Add 25 uL of TEMED tetramethylethylenediamine to the solution Swirl gently to mix Be careful to avoid introducing air bubbles WARNING TEMED Tetramethylethylenediamine is a flammable liquid and is extremely corrosive Vapors may travel considerable distance to sources of ignition and flash back TEMED is harmful by inhalation contact with the skin and if swallowed Use only in a chemical fume hood When handling wear lab coat safety glasses and chemical resistant gloves Immediately pour the gel Allow the gel to polymerize for at least 2 hours before use 5 0 Long Ranger The protocol for making a non denaturing 8 1 Long Ranger gel solution for SSCP Gel for SSCP differs from the
146. a new file name to preserve the original information To save a modified file under a new name Step Action 1 Open the File menu and choose Save As 2 Enter a name for the modified file and select a storage location for it 3 Click Save To save a backup copy of the file you are working with Step Action 1 Open the File menu and choose Save a Copy In 2 Enter a name for the file and select a storage location for it 3 Click Save A backup copy of the file is saved with the new file name and the original file remains on the computer screen Data Collection Software 9 57 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Archiving Files Gel Files Sample Files Printing Files Setting Up the Page for Printing Printing Procedure A gel file is automatically created for each run and contains the raw data collected by the data collection software Gel files typically require 10 70 MB of disk space Because gel files are so large they can be deleted from the hard disk once satisfactory sample files have been generated It is typically not necessary to save gel files once the tracking has been verified or adjusted and sample files are created See page 9 60 To archive Gel files use magnetic tapes removable cartridge drives CD ROMs or optical drives Gel files are too large to fit
147. a row select the row and click Delete Row If Then you are finished click OK to save your changes or Cancel to quit without making any changes you wish to change more preferences open the Page pop up menu and select another Preference 5 20 Setting Preferences Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Making Matrix Files for GeneScan Chapter Contents In this Chapter The following topics are discussed in this chapter Topic See page Terminology 6 1 Purpose of the Matrix File for GeneScan Applications 6 2 Why Matrices Must Be Remade 6 2 Evaluating Matrix File Quality 6 3 Creating a Matrix File for GeneScan Applications 6 6 Checking the Quality of a New Matrix File 6 8 Terminology Matrix vs The terms matrix file and instrument file are sometimes used interchangeably In this Instrument Files chapter the term Matrix file refers files that must be created to properly analyze data for GeneScan analysis software applications Instrument file refers to the files that must be created to properly analyze data for sequencing applications Making Matrix Files for GeneScan 6 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Purpose of the Matrix File for GeneScan Applications Purpose While the most intense fluore
148. ace of the plates It manifests itself as a band of little or no signal across the entire width of the gel image It usually occurs between 140 to 200 base pairs and typically lasts the equivalent of 20 to 40 base pairs Following this band signal strength usually returns to normal Glass plates can be cleaned manually or in a dishwasher The use of a laboratory dishwasher with a hot 195 F 90 C deionized water rinse cycle has been found to effectively remove suspect contaminants thereby eliminating any temporary loss of signal Gasket marks can be removed by cleaning the plates with an alcoholic KOH wash See The Gasket Mark on page 2 5 and Removing the Gasket Mark on page 2 9 We strongly recommend cleaning glass plates in a laboratory dishwasher with a hot 195 F 90 C deionized water rinse cycle Using a dishwasher helps ensure plates are cleaned effectively and consistently every time and will also eliminate the sporadic temporary loss of signal that can occur on this instrument described above Deionized water is required for the rinse cycle only Dishwasher recommendations are listed on page 2 8 When using a dishwasher we recommend you Connect the dishwasher to a high grade deionized water source Clean plates as soon as possible once the gel is removed Rinse residual gel from plates before loading in the dishwasher Initially use the longest deionized water rinse option on the dishwasher followed
149. ad region The number of channels in the read region are 194 for the standard ABI PRISM 377 and 377 18 instruments numbered 0 to 193 388 for ABI PRISM 377 XL instruments numbered 0 to 387 480 for ABI PRISM 377 96 lane instrument numbered 0 to 479 The type of comb used determines the number of channels per lane For example one lane of a 36 well comb is equivalent to approximately 5 channels Instrument Operation 3 47 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Manually Controlling the ABI PRISM 377 Instrument Procedure In manual control mode the instrument settings can be changed in real time The instrument must be idle before you can control it manually because commands sent via standard operation mode from a run sheet override commands entered manually Functions specified from the Manual Control function pop up menu are executed when the Execute button is clicked Functions continue running until The function is cancelled Anew function is selected To manually operate the instrument Step Action 1 Open the Window menu and choose Manual Control 2 Open the Function pop up menu Fxn Name and select a function Function pop up menu Em E Manual Control Fxn Fxn Name Yalue Range Ale Electrophoresis On No Value No Range Electrophoresis Off Electrophoresis Volts Madi Electrophoresis Current fia Electrophoresi
150. al Cooling System 22 0 eect ete e ene neee B 1 Hardware and Software Requirements 0 00 0 eee eect eee ene B 2 How the External Water Bath Functions 0 0 00 cece cece eee eee B 3 Installing the External Water Bath 20 cece ene B 5 Operating at Subambient Temperatures 0 0 00 eee eee eee B 6 Avoiding Condensation Inside the Instrument 0 0 0 0 eee e eee eee eee B 7 vii Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com C Parts and Accessories 2 cs ce cee cence esc eee ee Cul Appendix Contents eesse weccded eae ees Ged dette see dea eed ate al ae C 1 Part Number Updates sisii oi sesetsddcacdistessnee a bieraceaseacliws Sey hel E aoe dd ned eae C 1 Glass Plates Spacers and Clamps 00 cece eee eee eee ene e een eens C 2 Combs and Overlays seise sienes aea Ober bea p bes ha deb E wie Sebel EEE E C 3 Gel Cassette and Pouring Fixtures 0 0 0 eee cen eee eens C 3 Buffer Chambers and Gasket Kits 0 0 eee cece tenes C 3 Electrophoresis Cable and Electrode Assemblies 0 0 0 0 e eee eee eee C 4 Front Heat Transfer Plate 2 0 ec cence eens C 4 Two Pitch Eight Channel Loader Suppliers 0 00 00 ccc eee ee eee C 5 User s Manual 2 ssi5 04d esg s tte tae Seth a Sud ae Bee a ada eee E A eee C 7 ABI PRISM DNA Fragment Analysis Kits and Reagents 00 0000 e eee C 8 D Applied B
151. al should be less than 1 00 Note For virtual filter set C Green under Blue the second box from the top in the first column on the left is sometimes slightly above 1 00 This is acceptable 6 8 Making Matrix Files for GeneScan Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Check matrix quality as follows Step Action 1 From the Project containing your matrix standard Sample files open the Analysis Control window In the Analysis Control window select the colors for each sample Analyze O Print Results Sample File HS P2 1 copy v Analysis Control Print setup Size Standard P Parameters Created Thu Jul 18 1996 2 34 PM o20p27 2 1 HS P27 Standard GS 350 lt Analysis Parameters gt ef osep27 3 1 HS P27 Standard GS 350 K lt Analysis Parameters gt ef o4ep27 4 1 HS P27 Standard 6S 350 gt lt Analysis Parameters gt I e o5sF27 5 1 HS P27 Standard 65 350 lt Anatysis Parameters gt Select the four matrix standard Sample files Choose Assign New Matrix in the Project menu Select the matrix file Select numbers 1 2 3 and 4 on the left side of the window to highlight the colors for each row Use the Set Analysis Parameters dialog box in the Settings me
152. alling 9 44 to 9 45 list of modules 9 47 modifying and creating modules 9 45 to 9 46 naming conventions 9 42 to 9 43 standard modules 9 42 unused modules 9 46 viewing module settings 9 43 to 9 44 quitting Data Collection program 9 59 reinstalling ABI Prism 377 software 8 25 setting up forarun 3 19 shipped with instrument system file about 8 29 system software 9 2 1 21 1 22 1 11 software manuals part numbers C 7 solvents organic and glass plates 2 7 spacers cleaning 2 8 part numbers C 2 placement of 2 11 special text usage 1 5 SSCP avoiding condensation B 7 Chiller modules description of 9 44 dew point table B 7 hardware and software required B 2 to B 3 how external bath water works B 3 to B 4 installing external bath water MDE gel protocol A 18 performing a run B 6 purpose of external cooling system B 1 staining wells 3 35 status lights on instrument Status window description of 3 42 viewing gel and run temperatures 3 36 Status window description of 9 53 stock solutions storing A 23 stop and analyze feature using 3 40 storing instrument files 7 19 subambient temperature operation avoiding condensation B 7 Chiller modules description of 9 44 hardware and software required B 2 to B 3 how external water bath works B 3 to B 4 installing external water bath B 5 MDE gel protocol A 18 performing a run B 6 purpose of external cooling system B 1 B 5 3 8 suppliers for gel solutions A 22 for two
153. alysis Electropherograms can be printed on a color printer You can display the raw and analyzed data on the computer screen or edit and print it at any time Both types of analysis software are briefly described below ABI PRISM GeneScan Analysis Software automatically analyzes data collected from each GeneScan Analysis sample This software allows you to Software 4 Create custom analysis parameters or use the default analysis settings Size and quantitate PCR and other DNA fragments Automatically identify all DNA bands compare the mobility of each band to that of an internal lane standard and size the bands based on the sizing curve of the internal lane standards Interactively confirm and fine tune data analysis Display the results of an experiment as an electropherogram reconstructed gel image tabular data or a combination of electropherograms and tabular data Adjust the dye intensity of each sample individually or collectively Customize colors to more easily view large quantities of data Organize analyzed data in a variety of ways to fit the needs of your individual projects For example every project file can contain sample data from one or more runs Refer to the ABI PRISM GeneScan Analysis Software User s Manual for more detailed information 1 18 About This Instrument Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com ABI PRISM DNA Sequencing Ana
154. amine terminator kit 3 3 E L external cooling system lane tracking affects of air MDE gel protocol 3 18 bubbles 3 8 Long Ranger gels F 10 ammonium persulfate 3 15 4 75 Long Ranger Singel 3 16 5 Long Ranger gel for SSCP 3 17 not for SSCP 3 16 to 3 17 G gel solution ingredients 3 15 gel recipes recommendations 3 2 chemical abbreviations used 3 9 run conditions for 48 and 36 cm formamide deionizing 3 10 factors that affect read gels 3 15 lengths 3 4 list of recipes and M recommendations 3 2 MDE gel Long Manger gels list of gel recipes and 10 ammonium g p i ersulfate 3 15 recommendations p protocol 3 18 4 75 Long Ranger Singel 3 16 5 Long Ranger gel for SSCP 3 17 not for SSCP 3 16 to 3 17 ingredients and run P conditions 3 15 PAGE PLUS gels MDE gel for SSCP 3 18 gel recipes and PAGE PLUS gels recommendations 3 2 ingredients and run ingredients and run conditions 3 13 conditions 3 13 protocol 3 14 protocol 3 14 Polyacrylamide gels Polyacrylamide gels gel solution protocols 3 19 to gel formulation 3 2 3 21 gel solution protocols 3 19 to ingredients and run 3 21 conditions 3 11 ingredients and run protocol 3 11 to 3 12 conditions 3 11 sequencing chemistries 3 3 protocol 3 11 to 3 12 supplier information 3 22 polymerization gels APS reagent purity and factors that affect gel quality 3 4 associated problems 3 5 to 3 8 how affects gel quality 3 7 age of the gel initiator concentration 3 8 mobility sample inconsis
155. an accident misuse or neglect b modified or repaired by a party other than Applied Biosystems or c used in manner not in accordance with the instructions contained in the Instrument User s Manual This Warranty does not cover the customer installable accessories or customer installable consumable parts for the Instrument that are listed in the Instrument User s Manual Those items are covered by their own warranties Applied Biosystems obligation under this Warranty is limited to repairs or replacements that Applied Biosystems deems necessary to correct covered defects or failures of which Applied Biosystems is notified prior to expiration of the Warranty Period All repairs and replacements under this Warranty shall be performed by Applied Biosystems on site at the Customer s location at Applied Biosystems expense No agent employee or representative of Applied Biosystems has any authority to bind Applied Biosystems to any affirmation representation or warranty concerning the Instrument that is not contained in this Warranty Statement Any such affirmation representation or warranty made by any agent employee or representative of Applied Biosystems shall not be binding on Applied Biosystems Applied Biosystems shall not be liable for any incidental special or consequential loss damage or expense directly or indirectly arising from the purchase or use of the Instrument Applied Biosystems makes no warranty whatsoever with regard
156. an window Sometimes contaminants will migrate out of the read region Instructions for performing a plate check and determining which lanes to skip are listed in Chapter 3 Instrument Operation The scan window and the run sheet are described in more detail in Chapter 3 Instrument Operation and in Chapter 9 Data Collection Software A prerun is typically performed after the plate check and prior to starting the run The purpose of the prerun is to Equilibrate the gel temperature before samples are loaded Allow the operator to ensure the system is working properly During a prerun the pump heating system and scanner are activated Prerunning the gel is particularly important when performing a high speed 2400 scans hour run The gel must be at run temperature the temperature at which the samples will be electrophoresed when the samples are loaded to ensure appropriate denaturation conditions exist Prerunning also removes mobile ions from the gel and prevents extrusion gel oozing from between the plates from occurring when the run begins and a higher voltage is applied To perform a prerun the gel and all other necessary parts must be installed inside the instrument and a prerun module must be selected on the run sheet You start the prerun by clicking the PreRun button on the run sheet During the prerun you open the Status window shown below to monitor instrument conditions including the gel temperature
157. and Hot Start PCR Problems with primer choice concentration or degradation Refer to the troubleshooting guide in the GeneScan Reference Guide Inconsistent yields with control DNA Combined reagents not spun to bottom of PCR sample tube Place all reagents in apex of tube and spin briefly after combining Combined reagents left at room temperature or on ice for extended periods of time encouraging mispriming and other primer artifacts Put tubes in block immediately after combining reagents Combined reagents not thoroughly mixed Primers not uniformly suspended before adding to reaction mixture Primers can aggregate and settle to the bottom of the tube Vortex all primers reagents and reaction mixes minus enzyme thoroughly to ensure uniform concentration Pipetting errors Follow all these precautionary measures Calibrate pipettes Attach tips firmly Check pipetting technique Minimize pipetting small volumes for example make master mixes You may also want to consider using a 2 uL or other high precision pipette Yield gets progressively poorer for successive PCR amplifications performed over time Expired or mishandled reagents Check expiration dates on all reagents If not expired verify that reagents are being stored and used according to manufacturer s instructions Compare with PCR performance using fresh reagents Poor yield for multiplex PCR
158. apter 5 for instructions and more information 9 14 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Name of Field Description Instrument File Also referred to as a matrix file an instrument file is a set of up to three mathematical matrices used to compensate for the spectral overlap that occurs between the dyes used together as a set During system installation the engineer installing the system creates a default instrument file that is given the instrument serial number as a file name Depending on the chemistries used you may have to create additional instrument files An instrument file must be selected for data to be analyzed automatically at the end of a run For more information see About Automatic Data Analysis on page 9 38 Chapter 6 Making Matrix Files for GeneScan The chemistry kit protocol Automated DNA Sequencing Chemistry Guide P N 4305080 Comments Any additional information General Sample The following general information will help you prepare sample sheets Sheet Information Sample sheet information is used by the software to identify each individual sample when Sample Files described on page 9 60 are generated after a run Therefore it is critical that each sample name be entered in the field whose number corresponds to the gel lane in which the sample is loaded If
159. as the glass plates If the spacers are too long the gel injection device may leak Trim them to the proper size if necessary If the plates have never been used before read Before Using New Glass Plates on page 2 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Procedure To mount glass plates in the gel cassette Step Action 1 Place the cassette on a level clean surface 2 Raise the laser beam safety bar and turn all the clamps to the open position Clamp in closed Clamp in open Laser beam safety position position bar raised Bottom of cassette Check the inside surface of both plates for water droplets dust lint or anything else that might fluoresce or scatter light Clean the plates with a damp Kimwipe if necessary Place the rear plate in the cassette with the notched end oriented toward the bottom of the cassette IMPORTANT To avoid difficulty pouring the gel always load the front and rear plates with the same side of the glass facing out Refer to Before Using New Glass Plates on page 2 5 for more information Place the spacers on the rear plate as shown below notched side facing the middle of the plate Two to three droplets of water can be applied to the edge of the glass where the spacers will rest to keep them from mo
160. at a lower temperature or voltage 4 4 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Observation Gel image contains green blue streaks throughout run Blue or green streaks curtain at top of gel image Possible Causes Fluorescent contaminant in gel Recommended Actions Vacuum filter gel solution Cast gel in dust free environment Urea crystals present in gel Use room temperature reagents Pour at 20 23 C IMPORTANT Do not refrigerate Particles on outer surface of plates in read region Buffer leak Wipe read region with damp lint free Kimwipe Make sure that the plates are clamped correctly and that the upper buffer chamber gasket makes a proper seal Do not spill buffer behind the upper buffer chamber Wicking can occur Blue or green curtain obscuring entire gel image Warped gel plate Use gel plates from Applied Biosystems Green streak through entire gel lane Protein in template Clean up the template before performing sequencing reactions Greenish yellow haze Poor gel plate alignment Remove the gel plates and realign them correctly Fluorescent contaminant in gel Use fresh reagents Do not write on gel plates with marking pens Residual detergent on plates Rinse plates thoroughly with hot deionized water Troubleshooting 4 5 Artisan Techn
161. ata points analyzed is the same for each matrix standard Choose starting points for each sample such that all peaks are less than 4000 RFU and that both the starting and ending points have flat baselines and no peaks Making Instrument Files for Sequencing 7 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Making the Taq To make the Taq Terminator Matrix Terminator Matrix Step Action 1 In the Data Utility application choose Make Matrix from the Utilities menu The Make Matrix dialog box appears 2 In the Make Matrix dialog box click the Taq Terminator Matrix button at the lower left 3 Click on the box for each nucleotide base and enter the data file that corresponds to the correct matrix standard as shown in the table below Taq Terminator Box Matrix Ci dROX A dR6G G dR110 T dTAMRA IMPORTANT The order of matrix standard data files is different from that in the Dye Primer Matrix see Table 7 1 on page 7 10 4 Enter the same numbers for each matrix standard sample in the Start at and Points boxes as were used for the Dye Primer Matrix 5 Click Update File A dialog window appears 6 Choose dRhod_BigDye from the ABI folder within the System folder and click Open The Make Matrix dialog box should look like that shown below Note The numbers in the Start at and Points boxes are
162. ated prior to the change Verify that the Auto Analyze boxes are selected for each non matrix standard sample Figure 3 5 on page 3 27 Selecting Auto Analyze allows you to select analysis parameters a size standard and the auto print option If matrix standard samples are being run deselect Auto Analyze for all matrix standard samples If not already selected or if you wish to change the preference settings set the remaining fields as follows a Open the Analysis Parameters pop up menu and select an analysis parameters file Analysis parameters must be set up in the analysis software program prior to completing the run sheet Refer to the ABI PRISM GeneScan Analysis Software User s Manual for instructions b Open the Size Standard pop up menu and select the appropriate size standard file c Select the Auto Print boxes for analyzed data to be printed automatically Note If the pop up menus list lt none gt only software cannot find the folders that contain the analysis parameters and size standard files To correct this see Setting Folder Location Preferences on page 3 51 Proceed to Starting the Run or Closing the Run Sheet on page 3 34 Instrument Operation 3 33 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Auto analyze deselected This automatically deactivates the analysis parameters size standard and auto pr
163. atrix Name of file containing C data Start at Name of file containing A data Start at Name of file containing G data Start at Name of file containing T data Start at Points Name of new matrix file Dye Primer Matrix Tag Terminator Matrix Cancel OT Terminator Matrix Note The files you select for the four nucleotides are the sample files you named on the Sample Sheet when you electrophoresed the matrix standards 3 Specify the sample file to be used for each standard a Click the C button In the directory dialog box that appears select the file that contains the data from the C standard then choose Open to close the dialog box b Repeat this selection process with for the A G and T standards 7 20 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To add or replace a matrix in an existing instrument file continued Step Action 4 In each Start at text box leave the default as is or enter a different value Note The defaults of 2000 for start point and 1500 for data points to be used for the matrix are almost always appropriate Refer to the ABI PRism DNA Sequencing Analysis Software User s Manual for information on how to determine values for the Start at and Points boxes In the Points text box leave the default value as is or enter a differ
164. attire full length laboratory coat protective glasses gloves etc when handling and mixing hazardous chemicals Always work under a chemical fume hood when handling and mixing hazardous chemicals The room in which you work must have proper ventilation and a waste collection system Using a P 5000 Pipetman or equivalent add 5 mL of deionized water to the tube Vortex until all crystals dissolve The 10 APS solution can be stored in tightly capped tubes for up to 1 month Artisan Technology Group Gel Recipes A 15 Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 4 75 Long Ranger Follow the manufacturer s instructions to prepare the 4 75 Long Ranger Singel gel Singel solution 5 0 Long Ranger For SSCP applications follow the instructions listed under 5 0 Long Ranger Gel for Gel not for SSCP SSCP on page A 17 IMPORTANT The 5 0 Long Ranger FMC Corporation P N 50610 gel solution is hazardous waste Dispose of the unused working solution in accordance with all applicable federal state and local health and safety regulations After you finish pouring the gel thoroughly clean all work surfaces with water To prepare a 36 cm 5 0 Long Ranger 6 M urea gel Step Action 1 Instructions for preparing glass plates and two gel pouring methods are located in Chapter 2 Pouring Gels Glass plates and all other gel pouring equipment must be
165. ber C 4 removing and cleaning 3 45 Index 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com G gasket mark description of 2 5 how toremove 4 31 gel cassette cleaning afterarun 3 45 electrical shock hazard warning 3 9 how to carry with glass plates 3 4 how to carry with plates 2 10 installation procedure 3 8 to 3 10 loading gel plates into cassette 3 6 to 3 7 part numbers C 3 removing from instrument gel extrusion description 4 29 preventing 3 35 solutions for 4 29 gel files archiving 9 58 description of saving 8 23 using the GelDoc utility gel injection device fixture gel pouring methods overview of methods 2 4 plates mounted in cassette 2 10 using unmounted plates 2 20 gel quality factors that affect importance of 2 3 gel recipes chemical abbreviations used A 9 factors that affect read lengths A 4 list of recipes and recommendations A 2 Long Ranger gels 10 ammonium persulfate A 15 4 75 Long Ranger Singel A 16 5 Long Ranger gel for SSCP A 17 not for SSCP A 16 to A 17 ingredients and run conditions A 15 MDE gel for SSCP A 18 PAGE PLUS gels ingredients and run conditions A 13 protocol A 14 3 45 1 17 9 58 1 19 2 14 2 1 Index 4 Polyacrylamide gels gel solution protocols A 19 to A 21 ingredients and run conditions A 11 protocol A 11 to A 12 See Appendix A 2 1 sequencing chemistries A 3 supplier information A 22 gel recommendations
166. bles are trapped between the comb and the gel solution carefully remove and reinsert the comb until the bubbles are gone Follow these guidelines for consistent results To ensure complete polymerization wait a full two hours after casting the gel before use Use gels within two to six hours after casting After six hours resolution begins to noticeably deteriorate Gels that stand overnight can show significantly slower DNA migration due to the slow hydrolysis of urea to ammonium carbonate Since the amide groups of the polymer slowly hydrolyze into acid groups gels that stand 48 hours can also show significant loss in resolution beyond 350 bases Also the exposed areas top and bottom of the thin gels commonly used with the ABI Prism 377 DNA Sequencer dry out quickly Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Chemical Abbreviations Used Chemical Chemical abbreviations used in the following gel recipes Abbreviations Used Abbreviation Chemical Substance APS ammonium persulfate EDTA ethylenediaminetetraacetic acid TEMED N N N N tetramethylelthylenediamine TBE tris borate ethylenediaminetetaacetic acid buffer Preparing TBE Buffer 10X TBE To make 50 mL of 10X TBE Step Action 1 To a 50 mL screw cap tube add the following 5 4g Tris 2 8 g Boric acid 0 4g Na EDTA Distilled deionized water
167. buffer chamber quick disconnect Rear valves Front heat transfer plate heat transfer ground connection plate High voltage connection for lower buffer chamber Power switch and status lights Positioning pins Figure 3 1 Electrophoresis chamber 3 8 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Installation WARNING ELECTRICAL SHOCK HAZARD Severe electrical shock can result if Procedure you defeat the safety interlocks located in the door of the instrument Close the panel door before operating the instrument To install the gel cassette and lower buffer chamber Step Action 1 Open the right front panel of the ABI PRISM 377 instrument 2 Inspect the front and rear heat transfer plates and the two positioning pins inside the electrophoresis chamber Figure 3 1 on page 3 8 for residue If dirty clean with a Kimwipe CAUTION TBE crystals and dried acrylamide can accumulate on the positioning pins and heat transfer plates The presence of these residues can result in arcing Arcing is a luminous low voltage high current electrical discharge that can severely damage the instrument Note TBE crystals and dried acrylamide can also cause misalignment of plates resulting in a colored haze over the gel image 3 Place the lower buffer chamber on the bottom shelf of the electrophoresis chamber and connect the electrode cable to the
168. buffer to remove urea WARNING Tris borate EDTA TBE buffer can be harmful if inhaled ingested or absorbed through the skin It is irritating to the eyes skin and mucous membranes Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Optional Load formamide blue dextran loading solution in the well to the left of the first sample lane and in the well to the right of the last sample lane the comb creates additional wells on either side of the numbered wells This helps focus the bands in the first and last lanes WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Wash thoroughly after handling formamide Wear appropriate protective eyewear clothing and gloves Obtain a copy of the MSDS from the manufacturer If using a Then square tooth comb load one sample per lane into each consecutive well Refer to your protocol for the load volume or to Suggested Load Volumes on page 3 39 Change the pipet tip for each sample shark s tooth comb select Load Method 1 or 2 below Load Method 1 Load Method 2 50 of lanes used 50 of lanes used Refer to your protocol for the load volume or to Load one sample into Suggested Load Volumes on page 3 39 Change every other well the pipet tip for each sample Refer to your protocol a Load one sample into each odd numbered well for t
169. cations Preference Guidelines are provided in the tables below The information listed reflects the default folder names and locations as assigned automatically when ABI PRISM Data Collection Software is installed IMPORTANT To avoid problems setting up sample and run sheets we strongly recommend not changing the names or locations of these folders Instructions for setting Folder Location Preferences are listed on page 9 6 For more information see Data Collection Software Files and Folders on page 9 3 and Chapter 5 Setting Preferences Sequencing Sample Sheet Pop Up Menus Folder Location Preference Dialog Box Field Folder Name and Location DyeSet Primer Settings Folder ABI folder inside the System folder Instrument File Settings Folder ABI folder inside the System folder Run Sheet Pop Up Menus Folder Location Preference Dialog Box Field Folder Name and Location Plate Check PreRun and Run Modules Module Folder Modules folder or Chiller Modules folder if appropriate inside the ABI PRISM 377 folder Sample Sheet Sample Sheet Folder Sample Sheets folder inside the ABI PRISM 377 folder Instrument File sequencing only Settings Folder ABI folder inside the System folder Gel s Matrix File and Matrix File GeneScan only Settings Folder ABI folder inside the System folder Analysis Parameters GeneScan only GeneScan Analysis Para
170. ces Folder Location Preferences are set during system installation If files or folders are renamed or moved after installation data collection software may not be able to locate the firmware image file when necessary If this occurs a dialog box asking Where Is ABI Prism 377 Firmware is displayed S Firmware Image v lt 4 Folder containing Firmware file D ABI Prism 377 Firmware FEE Firmware file Where Is ABI Prism 377 Firmware Follow the procedure listed below to locate and download the firmware Refer to Chapter 5 Setting Preferences for more information Procedure To locate and download the firmware Step Action 1 Open the pop up menu in the dialog box and locate the folder containing the firmware image file Unless changed after system installation the filename is ABI Prism 377 Firmware It is located in a folder named Firmware Image inside the ABI PRISM 377 folder 2 Click Open Software automatically downloads the file to the instrument Firmware Image Sending Firmware Image to Instrument 3 Once the file is downloaded the following alert box is displayed Click Quit Quit application and relaunch for this A action to take effect 4 Double click the ABI Prism 377 Collection icon to launch the data collection software 4 24 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE
171. ch sample individually Instrument Operation 3 25 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Specify the Size To specify the size standard dye color Standard Dye Color Step Action 1 For all non matrix standard samples designate the color of the size standard by clicking in the box to the right of the appropriate letter in the Color column B blue G green Y yellow R red When selected a diamond is displayed in the Std column This must be specified for data to be analyzed automatically Sample Name Project Name Color Std Pres Size standard dye color is red Leave the Std column blank for matrix standard samples If necessary remove diamonds for matrix standard samples by clicking on the diamond Specify the Dyes Run To specify the dye colors run together in each lane in Each Lane Step Action 1 Click in the appropriate boxes in the Pres present column to select the dye colors that will be run in each lane for each sample Codes for the colors are displayed in the Color column B blue G green Y yellow R red Dyes must be specified for data to be analyzed automatically Click in these boxes to indicate the dyes being run in each lane To deselect a color click in the appropriate box to remove the X Enter Sample Info The information in the Sample Info and Comment
172. cintosh SAM version 4 0 or greater Norton Utilities Separate disks for each software program are included Store these disks in a safe place Installation The following general procedure can be used to reinstall ABI PRISM 377 instrument Procedure software such as sequencing or GeneScan analysis software More specific instructions for downloading and installing data collection software from our website are listed under Downloading Data Collection Software from Our Website on page 8 26 To install ABI PRISM 377 software Step Action 1 Quit any currently running applications 2 Hold down the Shift key and choose Restart from the Special menu to restart the computer with extensions turned off Continue holding down the shift key until you know the extensions are disabled Some Macintosh s will display a message that extensions are turned off All Macintosh s display extension icons that are X d over IMPORTANT If extensions are not turned off you may not be able to complete the installation Insert the Install disk into the appropriate drive If the Installer window does not open automatically double click the disk icon Open the Readme file and read it Double click the Installer icon Respond to the prompts as appropriate Click Quit when installation is complete OJON OAOA OQ Restart the computer to turn the extensions back on and rebuild the deskt
173. cludes GeneScan 350 Internal Lane Size Standard and loading buffer 402247 Kit B Amplified PCR Products Contains four tubes of pooled combined PCR products To generate the products each DNA sample CEPH 1347 01 1347 02 1347 10 1347 15 has been amplified with the same six fluorescent labeled PCR primer pairs in kit A All of the PCR products from one tube can be detected in one gel lane C 10 Parts and Accessories Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com ABI PRISM Linkage Mapping Set Version 2 50 Rxn Kits 300 Rxn Kits Panel Chromosome 403089 403118 Complete Set 1 22 X 403090 403119 1 1 403091 403120 2 1 403092 403121 3 2 403093 403122 4 2 403094 403123 5 3 4 403095 403124 6 3 4 403096 403125 7 3 4 403097 403126 8 5 6 403998 403127 9 5 6 403099 403128 10 5 6 403100 403129 11 7 8 403101 403130 12 7 8 403102 403131 13 9 10 11 403103 403132 14 9 10 11 403104 403133 15 9 10 11 403105 403134 16 9 10 11 403106 403135 17 12 13 403107 403136 18 12 13 403108 403137 19 12 13 403109 403138 20 14 403110 403139 21 15 16 403111 403140 22 15 16 403112 403141 23 17 18 403113 403142 24 17 18 403114 403143 25 19 20 21 22 403115 403144 26 19 20 21 22 403116 403145 27 19 20 21 22 403117 403146 28 X 450096 Individual Primer Pairs from the ABI PRism
174. comb in place one clear and one black Level raised support for example two empty pipet tip boxes Absorbent paper J J Gel injection fixture 2 Clear brace Black brace 2 14 Pouring Gels Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Attaching the Gel If you are pouring gels using unmounted plates proceed to Method 2 Pouring the Injection Device Gel Using Unmounted Plates on page 2 20 To attach the gel injection device Step Action 1 Place the gel cassette with plates on a level raised support The support s should be under the cassette not under the glass plates 2 Attach the black brace as follows a Hold the brace with the tabs pointing up Move the tabs up through the gap between the side rails and top bar of the cassette as shown below so the tabs rest on the rails Slide the brace along the rails and under the glass c Firmly push the brace under the plates until the tabs press against the rear plate Tabs on black brace EE Side rail Black brace in place with tabs firmly seated against rear plate Pouring Gels 2 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To attach the gel injection device continued Step Action 3 Attach
175. crylamide or acrylamide derivatives You can load up to 96 lanes on one gel depending on the model instrument you have The instrument is designed to accommodate different size gel plates so that the distance the sample migrates before detection can be varied This feature permits run times and sample resolution to be optimized according to the type of analysis either base calling DNA sequencing molecular sizing in base pairs or molecular quantitation Once the samples are loaded voltage is applied causing the fragments to electrophorese through the gel and separate according to size At the lower portion of the gel above the lower buffer chamber the fragments pass through an area called the read region see page 1 15 where a laser beam continuously scans across the gel The laser excites the fluorescent dyes attached to the fragments and they emit light at a specific wavelength for each dye The light is collected in 194 388 or 480 channels the 377 377XL or 377 96 respectively during each scan It is then separated according to wavelength by a spectrograph onto a cooled charge coupled device CCD camera so all four types of fluorescent emissions can be detected with one pass of the laser The data collection software collects the light intensities from the CCD at particular wavelength bands software selectable virtual filters and stores them on the Macintosh as digital signals for processing The collected data before it i
176. d interpeak baseline 6 4 e mail address for technical support 1 6 ergonomic hazard sample loading hazards and recommendations 3 37 error messages Macintosh operating errors 4 20 Execute button manual control mode 3 48 explosion warning nitric acid hazardous waste 4 32 exporting sample sheet information 9 27 external cooler off function 3 50 1 13 external cooler on function 3 50 external cooling system avoiding condensation B 7 hardware and software required B 2 to B 3 how an external water bath works B 3 to B 4 installing external water bath B 5 MDE gel protocol A 18 performing a run B 6 purpose of B 1 See also SSCP F Factura software description of File menu 9 10 files 1 21 archiving 8 23 9 58 deleting to ensure adequate disk space for new data files 3 40 gel files 9 56 9 58 Log file 9 54 opening and saving 9 57 temporary files created in System folder 9 8 firmware about the firmware 4 21 downloading new image 3 50 4 24 file location 3 51 symptoms of corrupt firmware 4 21 firmware file folder location and description of 3 51 fluorescent dyes 1 13 folder locations folder names and locations 3 51 to 3 52 how to set 3 52 to 3 53 setting preferences 5 5 to 5 7 table of locations 9 5 to 9 6 formamide chemical warning 3 3 deionizing A 10 formamide blue dextran using in empty wells 3 38 fragment analysis kit and reagent part numbers C 8 to C 11 front heat transfer plate installing 3 18 part num
177. damine Dye Terminators Color Base Dye Base Dye Blue C 5 FAM G R110 Green A JOE A R6G Yellow G TAMRA T TAMRA Red T ROX C ROX Raw Data Colors for Virtual Filter Set E dRhodamine Terminators BigDye Primers BigDye Terminators Color Base Dye Base Dye Base Dye Blue G dR110 C FAM dR110 G FAM dR110 Green A dR6G A FAM dR6G A FAM dR6G Yellow C dTAMRA G FAM dTAMRA T FAM dTAMRA Red T dROX T FAM dROX C FAM dROX Analyzed Data The Sequencing Analysis program converts the information collected by the data Color Guide collection program so that after analysis the colors representing each base are consistent regardless of the chemistry used The colors on all displays of analyzed data including printed electropherograms are as follows Color Guide for All Analyzed Data Base Color C Blue A Green G Blacka T Red a G is shown as yellow in AutoAssembler software Making Instrument Files for Sequencing 7 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Making an Instrument File for Virtual Filter Set A from Matrix Standards Summary of The following is a brief outline of the steps involved in making an instrument file Procedure What Matrix Standards are Required Running Matrix Standards to Obtain the Raw Data Step Action 1 Run the appropriate matrix standards for your instrument verify that lane tracking is correct and verify that peaks e
178. data transferred to BioLIMS is identified by the gel file name only The Project Info preference allows you to enter a project name information about the project in a comments field and a person s name as the project owner This information is transferred to the sample sheet when a project name is selected Setting Project Info To set or change Project Info preferences Preferences Step Action 1 If the Preferences dialog box Then is not open open the Windows pull down menu select Preferences and then select Project Info is open open the Page pop up menu and select Project Info Click Add Row to add a row for each project to be defined Preferences Page Project Info Y Project Hame Project Comment Project Owner Comments for Project 1 Jody Carrillo Project 3 Comments for Project 3 Jody Carrillo Project 2 Comments for Project 2 Jody Carrillo Add Row Delete Row Cooner Cor To define the first project select the first cell in the Project Name column and enter aname Press the tab or right arrow key to move to the Project Comment column and enter comments or leave the field blank Press the tab or right arrow key to move to the Project Owner column and enter an owner s name or leave the field blank Continue adding information in the same manner for the remaining projects To delete
179. detail below for academic purposes only The protocols contained in this appendix have already been optimized Therefore experimentation with these variables is not necessary Factors that affect the rate of polymerization Factor Affect on Rate of Polymerization Temperature Temperature directly affects the rate of polymerization The rate of polymerization determines the properties of the gel Therefore temperature control is crucial for the production of high quality gels Optimal room temperature for gel production and polymerization is 20 to 23 C The gel solution and glass plates should be the same temperature Gels produced within this temperature range are transparent They are not too porous and more elastic than gels produced in colder or warmer temperatures These gels are also the most reproducible Gels produced in cold environments such as 4 C are turbid porous and inelastic Run to run reproducibility is greatly compromised Gels produced in environments that are too warm are inelastic because the polymer chains are too short The result is non reproducibility from run to run Oxygen Oxygen acts as a free radical trap thereby inhibiting polymerization The result is a porous gel Therefore polymerization must be fast enough to prevent too much oxygen from dissolving into the gel solution during polymerization Follow these guidelines to minimize the amount of oxygen that dissolves in the gel s
180. dow displays the intensity of the fluorescent emissions being collected as samples pass through the read region of the gel and the dyes are excited by the laser The emissions are displayed as peaks Each dye virtual filter is represented by a different colored line The display is continuously updated during instrument operation The current scan number is displayed in the upper right hand corner of the window Raves Ginnie inane Window R194 Y 4783 C Freeze updates Current Scan Number 1088 Freeze Updates Current Scan Number 1088 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Instrument Operation 3 41 3 42 Instrument Operation Gel Window Like the Scan window data in the Gel window also represents the fluorescent emissions collected from the samples as they pass through the read region of the gel The first fragments scanned by the laser appear at the top of the window and move down as new data is collected Gel File Gel View 40 60 680 100 120 140 160 180 Status Window The Status window displays the current status of the instrument green boxes This information is updated approximately every three seconds Open the Status window by choosing Status from the Window menu The electrophoresis voltage gel temperature and laser power are setpoints specified by the module currently being executed Electrophoresis current and electrophoresis power ar
181. e Genotyper can convert GeneScan fragment data to called alleles Genotyper results can be transferred to databases for storage spreadsheets for statistical analysis or linkage analysis software GenoPedigree software is an interactive pedigree diagram editor that reads and writes its own documents containing pedigree layout and style information GenoPedigree can also generate files for linkage analysis GenBase software is a database application that stores data for genotypes pedigrees markers traits diseases and other relevant information You can import data from or export data to GenBase from Genotyper and GenoPedigree Primer Express software allows you to design analyze and order oligonucleotides The process is simple import or drag sequences into the application then select primers using the Automatic Find mode default settings or custom parameters settings The software performs the appropriate calculations and automatically updates all relevant views You can select target amplicon sequences automatically translate DNA sequences to amino acids exclude DNA sequences from an amplicon label exon intron junctions and annotate important sequence sites The software has specific ready to use templates for many PCR and sequencing applications TaqMan probe and primer design Standard nested allele specific multiplex and RT PCR Sequencing Cycle sequencing Batch processing of multiple DNA documents
182. e information refer to Chapter 6 Making Matrix Files for GeneScan 3 24 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Lane number on the gel Sample Name Sample A loaded in lane 1 Lane 2 left empty First matrix standard sample loaded in lane 3 Lane 4 left empty Matrix standard sample green Second matrix standard sample loaded in lane 5 Figure 3 4 Sample name column on a GeneScan sample sheet Select Project Names The Project Name field is for BioLIMS version 2 0 and up users only Otherwise the field can be left blank If a project name is not identified the data transferred to BioLIMS is identified by the gel file name only Project names must be defined prior to setting up the sample sheet as Project Info Preferences Refer to Project Information Preferences in Chapter 5 for instructions and more information To select project names Step Action 1 Open the Project Name pop up menu for each sample and select the project name Project Name Project 1 H Project 2 Project 3 aa Click here to open the Project Name pop up menu 2 If the Project Name is the same for all remaining samples click in the column heading to select the entire column open the Edit menu and select Fill Down Otherwise select the appropriate Project Name for ea
183. e Causes Recommended Actions The following dialog box is displayed when data collection software is launched Select Sample Sheet Folder ABI Prism 377 96 Colle Y Macintosh Ba Firmware Image D C Modules Runs Desktop 7 Sample Sheets Select Firmware Image Folder names have been changed Preferences file corrupt Reset the Folder Locations Preference Instructions are listed in Chapter 9 on page 9 39 and in Chapter 5 Setting Preferences Message indicating there is not enough room to store data from run Not enough room on the hard disk Delete files particularly gel files from the hard disk Gel files are large and are stored in the corresponding Run folder created for each run Data not analyzed automatically Sample Sheet not completed or completed incorrectly Complete Sample Sheet as described GeneScan Analysis Software Run Defaults set incorrectly in data collection program in the Preferences under the Window menu Make sure that the Autoanalyze box is checked and that the GeneScan Run Default appears with the correct path name Insufficient free RAM Restart the computer before collecting data Note You should always restart the computer before collecting data Conflicting extensions Choose Extensions Manager from the Control Panels Turn off any extensions that were not part o
184. e Select All to select all the files in the Chiller Modules folder Click and drag all the chiller module files into the Modules folder Close both folders 9 44 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Modifying and Creating Modules Setting Preferences to Access Chiller Modules Directly in the Chiller Modules Folder To set preferences to access chiller modules in the Chiller Modules folder Step Action 1 Open the ABI PRism Data Collection Software version 2 1 2 Open the Window menu select Preferences and then select Folder Locations 3 Click the Module Folder to open the Select Module Folder dialog box Select Module Folder ae C Chiller Modules COFirmware Image Co Modules Desktop C Runs C Sample Sheets Eject Cancel Select Chiller Modules Click once on Chiller Modules to highlight the folder Click the Select Chiller Modules button Click OK to save this setting IMPORTANT To run the instrument using standard conditions change the Module folder preferences setting from the Chiller Modules folder back to the standard Modules folder ABI PRISM 377 Data Collection Software version 2 1 module files can be Permanently modified Temporarily modified for one run Used as templates to create custom module files without changing the original modu
185. e active window 3 not open open a new run sheet as follows a Open the File menu and select New b Click either the Sequence Run or GeneScan Run icon as appropriate A new Run folder is created automatically in the Runs folder inside the ABI PRISM 377 folder Note We recommend using the same run sheet for the plate check prerun and run This will limit the number of run folders created per run to one 3 28 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Select the Plate To select the plate check prerun and run modules Check PreRun and Run Modules Step Action 1 Open the Plate Check Module pop up menu and select a plate check module L KUN br 24uu Plate Check Module DAQECIa Digs Plate Check B Plate Check C Plate Check D Run Module lt IMPORTANT If the pop up menus list lt none gt only software cannot find the folder that contains the modules See Setting Folder Location Preferences on page 3 51 2 Open the PreRun Module pop up menu and select a prerun module Open the Run Module pop up menu and select a run module Select the Number To select the number of lanes of Lanes Step Action 1 Open the Lanes pop up menu and select the appropriate value none For GeneScan applications For Sequencing applications Lanes e 24 Full Scan 24 34 36
186. e corrupt Below is a list of symptoms that can indicate corrupt firmware on the instrument and recommended corrective actions If any of the following symptoms occur perform a single reset See page 4 22 Flat scan lines or no scan lines when they should be present in the Scan window No response to commands from the computer If any of the following symptoms occur perform a total reset or cold boot as appropriate see pages 4 22 and 4 23 No response to commands from the computer even after performing a single reset Rear panel LEDs are stuck in one pattern e g all on or all off The computer cannot load a new image onto the instrument The computer appears to have downloaded a new firmware image onto the instrument however the instrument still does not run the rear panel LEDs remain stuck in one pattern Troubleshooting 4 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Performing a Single Reset Procedure Using the eraser end of a pencil or similar object press the red reset button on the back of the instrument once If communication is not restored perform a total reset as described below Performing a Total Reset What is a Total Reset When a total reset is performed the firmware currently installed on the instrument is erased Data collection software must be restarted so that a new copy of the firmware is loaded automatically onto the instrument
187. e emissions within each dye set Blue represents the shortest wavelength and red represents the longest The colors on the real time displays therefore represent the wavelengths of the dyes being detected rather than the bases being detected Colors Represent Different virtual filter sets use the same four colors to represent different wavelengths Relative so the colors do not represent actual wavelengths They represent the relative Wavelengths Wavelengths of the four dyes in each dye set For example Filter Set A uses the four colors to represent wavelengths within Dye Set 1 and Dye Set 2 Each of the chemistries used for preparing DNA is associated with a dye set Each dye set labels the four bases differently so the relative wavelength and therefore the color associated with each base varies with the chemistry used to label it Due to this the four colors on the real time displays represent different bases depending on the chemistry used for labeling The tables below describe the colors that represent each of the four bases on the real time displays 7 4 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Raw Data_ The following tables lists the raw data display colors and dyes for the gel image and Color Guide raw data based on the virtual filter set used Raw Data Colors for Virtual Filter Set A Fluorescein Rhodamine Dye Primers Rho
188. e in a well ventilated area On a routine basis thoroughly clean surfaces subject to contamination To inject the gel solution Step Action 1 Remove the clear brace and comb 2 Draw at least 35 mL of gel solution into a 60 cc syringe filling it slowly to avoid introducing air bubbles IMPORTANT Draw enough solution into the syringe to inject the entire gel without having to stop and draw more solution 3 Check the tip of the syringe The gel solution should be at the tip and no air bubbles should be present Adjust the solution and remove air bubbles if necessary 4 Screw the tip of the syringe into the luer fitting IMPORTANT Do not overtighten Overtightening will strip the threads Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To inject the gel solution continued Step Action 5 Slowly and steadily inject the solution between the plates At the same time constantly tap the top plate directly in front of the gel path to eliminate air bubble formation Injection takes approximately 60 seconds Stop when the solution fills the space between the plates Allow a small amount to pool in the well of the black brace CAUTION Tapping the plate with a tool such as a rubber mallet or reflex hammer can break the plates Note If you need to refill the syringe be careful not to introduce air through the fitting Keep excess solution i
189. e limits The instrument shuts down automatically if limits are exceeded Setpoints and limits are shown in the grey boxes Status Instrument State mar Running Laser Power m Running Electrophoresis Power mE On Door EET Closed Time Remaining 03 14 22 9 Total 03 30 00 Electrophoresis Electrophoresis Electrophoresis Gel Laser Voltage kY Current m Power W Temperature C Power mW Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Electrophoresis History Window The Electrophoresis History window displays the set and actual values for the electrophoresis power supply and gel temperature throughout the course of a run The scale for each panel is adjustable The information in the Electrophoresis History window is also stored in the Gel file To display the electrophoresis history window open the Window menu and choose Electrophoresis History Electrophoresis Histor Click here to adjust scale Scan numbers Instrument Operation 3 43 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Viewing the Log File A Log file is created for each run when the run is started Log files contain a comprehensive record of all error and status messages generated by data collection software during a run including Start and stop times of
190. e name and the location where the sample sheet will be stored is displayed a Change the file name now if desired b Click Save IMPORTANT Although it is an option we do not recommend changing the storage location of the sample sheet If the location is changed software will not be able to locate the sample sheet when you set up the run sheet Folder where sample sheets are stored Pd Sample Sheet 5 13 98 3 49 P Eject p Sample Sheet 5 13 98 4 20 P D Sample Sheet 5 14 7 Desktop u Sample Shee 5 157 M a fa Sample Sheet 5 15 98 3 34 PM g Save this document as Sample sheet file name Note You can also save the sample sheet by opening the File menu and selecting Save Save As or Save A Copy In Data Collection Software 9 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com About Sample Sheets for GeneScan Applications What is aGeneScan A GeneScan sample sheet shown below is a file that contains information used for Sample Sheet Sample identification and tracking Data analysis There are two types of sample sheets one for GeneScan and one for sequencing analysis applications The information entered on the sample sheet is imported to the GeneScan run sheet described on page 9 28 IMPORTANT Do not mix GeneScan and sequence analysis samples on the same sample sheet or in the same run
191. echnology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Selecting Project The Project Name field is for BioLIMS software users only Otherwise the field can be left blank If a project name is not identified the data transferred to BioLIMS is identified by the gel file name only Names and Entering Comments Project names must be defined prior to setting up the sample sheet as Project Info Preferences Refer to Project Information Preferences in Chapter 5 for instructions and more information To select project names and enter comments Step Action 1 Open the Project Name pop up menu for each sample and select the appropriate project name Project Name e Project 1 Click here to open the See Project Name pop up menu 2 If the Project Name is the same for all remaining samples click in the column heading to select the entire column open the Edit menu and select Fill Down Otherwise select the appropriate Project Name for each sample individually 3 To enter a comment click in the Comments field and type the information Saving and To save and close the sample sheet Closing the Sample Sheet Step Action 1 Do one of the following Open the File menu and select Close Click the box in the upper left hand corner of the window and then click Save Click Save A dialog box showing the default sample sheet fil
192. ect File Names is open open the Page pop up menu and select File Names For sample sheets run folders and run sheets a Enter the file or folder name desired in the appropriate field b Open the associated pop up menu to specify the date and time lt date gt or nothing lt none gt as a suffix Preferences Page File Names Type new names b Sample Sheet 4 m directly in these fields Sample Sheet Run Folder Type option 8 to Run Folder _ ater r make a bullet muh Run File Run adate gt vj Sample File d lt lane number w lt sample name gt Y Cancel ok Sample File suffix pop up menu lt none gt date serial number global serial number sample name gt none a alaha numbers Sample File prefix sample name gt pop up menu For Sample Files a To change the prefix open the prefix pop up menu and make a new selection b To change the suffix open the suffix pop up menu and make a new selection IMPORTANT Limit the number of characters in sample file names to 27 or less Do not use colons slashes or symbols in sample names Note The global serial number can be reset by changing the value in the General Settings Preferences window Refer to page 5 15 for more information If Then you are finished click OK to save your changes o
193. ect the dishwasher to a high grade deionized water source Clean the plates as soon as possible once the gel is removed Rinse residual gel from the plates before loading in a dishwasher Initially use the longest deionized water rinse option on the dishwasher followed by a drying cycle After some experimentation you may be able to reduce the rinse time Do not use a detergent Avoid excessive handling of dry plates with ungloved hands Recommended dishwashers are listed below If a dishwasher is not available follow the procedure listed in Chapter 2 Pouring Gels Recommended The following dishwashers have been found to work well A customized plate rack may Dishwashers be required Item Part Number Supplier Lancer 1600 dishwasher Lancer 1600 UP Lancer USA Inc with facility for drying 705 West Highway 434 Longwood Florida 32750 Telephone 407 332 1855 Lancer UK Ltd 1 Pembroke Avenue Waterbeach Cambridge CB5 9QR Telephone 44 01223 861665 Fax 44 01223 861990 Sequencing plate rack 50 SPR 16 Lancer USA Inc as listed above plate capacity for Lancer dishwasher Labconco Undercounter 15 352 801 Fisher Scientific SteamScrubber U S Headquarters Washer Dryer 585 Alpha Drive Pittsburgh Pennsylvania 15238 Customer Service 1 800 766 7000 Fax 1 800 926 1166 Internet http www fishersci com 8 4 System Maintenance Artisan Technology Group Quality Instrumentatio
194. ed Instrument For sequencing applications only A set of mathematical matrices used to compensate for the spectral overlap that occurs between the dyes used together as a set Sometimes referred to as a matrix file Matrix For GeneScan applications only A mathematical matrix used to compensate for the spectral overlap that occurs between the dyes used together as a set Data collection software uses files from both the ABI PRISM 377 and ABI folders For the preparation of sample and run sheets Toconirol the instrument and collect data As such data collection software must always know where the folders containing these files are located on the hard drive During system installation the Applied Biosystems Field Service Specialist specifies the location of these folders by setting the Folder Locations Preference to the recommended defaults Periodically you may have to reset these preferences Refer to Chapter 5 Setting Preferences on page 5 5 for more detailed information and instructions Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Setting Folder Location Preferences Overview To complete sequencing sample sheets and run sheets you must select files from various pop up menus If one of these pop up menus lists lt none gt only software cannot find the folder that contains the pertinent files To correct this you must reset the appropriate Folder Lo
195. ee eee ee eee 9 8 Menu Command ess asiera cae eee ecko dee a a are Se ee EN 9 9 About Sample Sheets for Sequencing Applications 0 0 0 ee eee eee eee 9 14 Preparing a Sequencing Sample Sheet 0 0 cee ee ee eee ee 9 16 About Sample Sheets for GeneScan Applications 0 0 eee ee eee 9 20 Preparing a GeneScan Sample Sheet 0 0 cece cece een eee 9 22 How to Enter Information on Sample Sheets 0 0 00 cece eee eee eee 9 26 Importing and Exporting Sample Sheet Information 0 0 0 00 0000 9 27 About Run Sheets cs sce wi tee ne daaadude set a ie Sesh Racha E hase bb eels eed 9 28 Preparing a R n Shete ssec scc ee eee eee eed bee eae bee Gee eae aie 9 31 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com About Automatic Data Analysis 0 0 eee eee e nen e eas 9 38 Commands Used to Perform a Plate Check PreRun and Run 0 0005 9 39 Modules riais ae e of deans drat a Seen AE ean 2S ae Seater old See inde eee 9 42 DyeSet Primer Files soci ecce ety ens ehea Sede eae wee NE athe Wee ae aera tee dente 9 49 Virt al Filter Sets cei eios e ang Sie Sa eai Sle Sais aS des ee R E eae wes 9 51 Viewing Data and Instrument Status 0 0 0 cee eee 9 53 Opening and Saving Files ces ccd casa bbe ee peed eee bee EERO RYE EE Deb 9 57 Archiving Pues pesei neret hele ha aR dea aba ew ee ane aged E yan a E AR AUSS 9 58
196. eed 888 88 SOURCE www artisantg com S safety warnings TEMED 3 6 3 23 urea 3 6 sequencing chemistries 3 3 SSCP MDE gel protocol 3 18 stock solutions storing 3 23 subambient temperature operation MDE gel protocol 3 18 suppliers for gel solutions 3 22 T TEMED effect of changing concentration 3 8 oxidized form 3 6 safety warning 3 6 3 23 storing 3 23 temperature how affects gel quality 3 8 tracking affects of air bubbles 3 8 Tris contaminants reagent purity and associated problems 3 6 U urea contaminants 3 6 effect of impurity 3 6 safety warning 3 6 WwW warnings acrylamide 3 5 3 23 bis acrylamide 3 5 3 23 TEMED 3 6 3 23 urea 3 6 rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com
197. eed to Verifying the Raw Data on page 7 7 to verify the raw data is satisfactory and to generate sample files 7 6 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Verifying the Raw After you run the matrix standards the next step is to verify that the run was Data successful and you have raw data for the matrix The following procedure requires you open gel files track and extract lanes and create and view sample files Refer to the ABI PRISM DNA Sequencing Analysis Software User s Manual for instructions on how to perform these operations To verify lane tracking and peaks in the raw data Step Action 1 Launch the Sequencing Analysis Software program 2 Open the gel file and make sure auto analysis is deselected 3 Track the matrix standard sample lanes and extract them into sample files IMPORTANT Because the Tracker program only recognizes red data you have to adjust the tracker lines by hand for the green blue and yellow standards Note The raw matrix data must be contained in sample files If you extracted the matrix data in BioLIMS mode export the data to sample files using the Sample2DB program For more information on exporting BioLIMS database records see the ABI Prism BioLIMS Sample2DB Software User s Manual P N 4304072 Open each sample file in a Sample window and verify tha
198. elect the appropriate file If one does not yet exist leave the field set to lt none gt A file must be selected for automatic data analysis IMPORTANT If the pop up menu lists lt none gt only software cannot find the folder that contains the instrument matrix files To correct this you must set the Settings Folder Preference to the ABI Folder See Setting Folder Location Preferences on page 3 51 Select the Run To select the run mode well to read distance and operator Mode Well to Read Distance and Step Action Operator 1 If this is Then a standard instrument proceed to the next step an XL or 96 lane upgrade instrument verify that the correct mode is displayed in the Run Mode field Lanes 96 Lane Scan XL Scan Run Mode Full Scan Open the Well to Read distance pop up menu and select the appropriate value Click in the Operator field and type your name 3 30 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Enter the Collection To enter the collection time Time Step Action 1 Enter the duration of the run in the Collect time field Use the run time recommended in your protocol The following are suggested run times only Suggested Collection Time in Hours Long Ranger Type of Run LR PAGE PLUS Well to Read Scans
199. en open the Windows pull down menu select Preferences and then select GeneScan Run Defaults is open open the Page pop up menu and select GeneScan Run Defaults 2 To enter an operator s name type the name directly into the Operator field Preferences Page GeneScan Run Defaults hd Operator wTR CET em Lares 34 PreRun Module GS PR 364 2400 w Run Module GS Run 364 2400 w C aAutoanalyze with Analysis Parameters Analysis Default w Gel s Matrix File D MODULE MATRIS w Size Standard GS 350 2400 36 5TD Y C Auto Print tance Cox 3 To change the default setting for the WTR distance Lanes PreRun Module Run Module Gel s Matrix File and Size Standard open the pop up menu for each parameter and select the desired default Setting Preferences 5 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 5 14 To change GeneScan run sheet preferences continued Step Action 4 To specify that data be automatically analyzed by GeneScan analysis software a b c d e Select the box labeled Autoanalyze with Open the autoanalyze pop up menu select Other and then select the appropriate analysis application ABI PRISM GeneScan Analysis Software Open the pop up menu for Gel s Matrix File and select the appropriate file Open the pop up menu for Si
200. en to remake 6 2 optimizing electrophoresis conditions 4 34 to 4 35 run sheets about 9 29 to 9 30 preferences setting 5 13 to 5 14 preparing 9 31 to 9 37 sample sheets about 9 20 to 9 21 entering information 9 26 importing exporting information 9 27 preferences setting 5 12 preparing 9 22 to 9 25 acrylamide denaturing gels described A 2 reagent purity and associated problems A 5 safety warning A 5 A 23 storing A 23 air bubbles affects on gel quality A 8 between comb and solution A 8 alcoholic KOH Wash procedure 4 30 Ammonium Persulfate See APS amplification troubleshooting guide 4 16 analysis parameters 3 28 3 33 3 34 3 51 3 52 5 14 9 31 9 36 automatic data analysis 9 38 folder location 5 2 5 5 5 6 9 5 analyzing data 1 18 3 46 See also automatic data analysis Apple menu about 9 9 Applied Biosystems warranty D 1 Index 2 APS effect of changing concentration A 8 storing A 23 archiving data from runs 8 23 to 8 24 gel and sample files 9 58 instrument files 7 19 recommendations for See Also saving arcing instrument damage warning 3 9 why it occurs 3 9 Auto Print checkbox 9 58 AutoAssembler software as part of GeneAssist 8 20 system 1 21 description of software 1 21 to 1 22 automatic data analysis analysis parameters 3 28 3 33 3 34 3 51 3 52 automatically printing results 3 32 3 33 deselecting 3 31 for GeneScan runs 3 33 for sequencing runs 3 32 B backing up data from runs 8 23
201. ent value See the Note in the preceeding step Click the Update File button In the directory dialog box that appears a Select the name of the instrument file to which you want to add the new matrix b Click Open Click the appropriate button for the chemistry Dye Primer Taq Terminator or T7 Terminator at the bottom of the Make Matrix dialog box IMPORTANT When you add a new matrix to an instrument file it overwrites any existing matrix of the same type Other matrices in the file are not affected Click OK to start the matrix calculation The calculation takes about one minute 10 When the message Make matrix successfully completed appears click OK or wait approximately 20 seconds for the dialog box to disappear If an error message appears and the matrix is not made see Correcting Errors in Matrix Creation on page 7 22 11 Verify the accuracy of the instrument file by following the appropriate procedure Making an Instrument File for Virtual Filter Set E from Matrix Standards on page 7 10 or Making an Instrument File from a Sample File on page 7 18 12 Store and backup the file as described under Storing and Backing Up the Instrument File on page 7 19 Making Instrument Files for Sequencing 7 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Correcting Errors in Matrix Creation
202. eous solution Preparation Procedure Step 1 2 3 4 A 18 Gel Recipes Use a 0 2 ym filter unit to vacuum filter the MDE solution containing glycerol and 1X TBE for 5 minutes IMPORTANT If the plates are not clean and mounted in the gel cassette or other device clean and mount them now before adding the polymerizing agents to the gel solution Instructions are listed in Chapter 2 Pouring Gels Adding the polymerizing reagents Step Action 1 With the filtered MDE solution at room temperature add 200 pL of 10 APS Swirl gently to mix Be careful to avoid air bubbles Add 25 uL of TEMED to the solution Swirl gently to mix Be careful to avoid air bubbles Immediately pour the gel Allow the gel to polymerize for at least 2 hours before use Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Polyacrylamide Gel Solution Protocols Preparing the To make 150 mL of 40 polyacrylamide stock solution use 40 Polyacrylamide Stock Solution Application Acrylamide g Bisacrylamide g SSCP 58 5 1 56 All other applications 57 3 Note For SSCP always use a low cross linker concentration 37 5 1 acrylamide bisacrylamide in the polyacrylamide stock solution For other applications typically use a 19 1 stock solution To prepare the 40 polyacrylamide stock solution Step Action 1
203. er Modules J Extensions Q Firmware Image fi Modules Old Modules oO Runs 7 Sample Sheets untitled folder Eject Folder located Select Modules and selected Click the Select field at the bottom of the dialog box to set the folder location Do not click Open Select Module Folder inni Chiller Modules c Extensions Eject Firmware Image old Modules enter N B sarr Sample Sheets Cuntitied folder 5l N elect Modules he Ma Click this field to set the location Return to step 2 to set additional folder location preferences or click OK if finished 9 6 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Data Flow Between the Computer and Instrument Overview of Data flows between the Macintosh computer and the ABI Prism 377 instrument as Data Flow shown below ABI Prism 377 Instrument Macintosh Computer Operating Sample gt instructions Gra A DATA m Run Sheet gt COLLECTION Module PROGRAM files i Electrophoresis Sequencing Analysis Raw sample data Gel File ysi ANALYSIS GeneScan PROGRAMS Analysis 7 Data collection software sends the operating instructions specified in modules selected on the run sheet to the instrument As the run is performed the instrume
204. er a hook at the right end of the chamber a Remove the flathead plastic screw b Lift the electrode assembly from the left side c Pull it from beneath the hook at the right end of the buffer chamber 3 Insert the new assembly into the buffer chamber If replacing the assembly in the lower buffer chamber be sure the assembly is properly situated under the hook at the right end of the chamber 4 Reinstall the screw s 8 12 System Maintenance Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Remove these screws The lower buffer chamber and lift the L shaped has a hook at the right end fixture out of the chamber Electrode fixture Electrophoresis cable Bpo Front of buffer chamber Instrument connection Figure 8 1 Cable and electrode assembly in the white upper buffer chamber P N 604078 Remove these screws and lift the L shaped fixture out of the chamber Electrophoresis cable A Front of buffer chamber Instrument connection Figure 8 2 Cable and electrode assembly in the transparent upper buffer chamber P N 4304406 System Maintenance 8 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com CCD Pixel Position Value Overview The charge coupled device CCD pixel position value is a reference point for alignment of the CCD camera with the lase
205. er a name for each matrix sample To help ensure a robust matrix is produced we strongly recommend you follow these guidelines Leave at least one empty lane between non matrix standard samples and matrix standard samples Leave one empty lane between each matrix standard sample v Lane number on the gel Empty lanes Data Collection Software 9 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Selecting the Different dye set primer files can be used for the same run as long as the virtual filter DyeSet Primer File set is the same for all samples Dye set primer file names for Rhodamine Terminators are similar to those for BigDye Terminators and can easily be mistaken for one another If the wrong file is selected base spacing in the data will not be noticeably affected C and T bases will be miscalled If you are not sure which file to select refer to the chemistry kit protocol and to DyeSet Primer Files on page 9 49 To select a dye set primer file Step Action 1 Open the DyeSet Primer pop up menu and select the appropriate file for the first sample You must select a file if you want the data to be analyzed automatically at the end of the run IMPORTANT If the pop up menu lists lt none gt only software cannot find the folder that contains the dye set primer files To correct this you must set the Settings Folder Preference
206. er of lanes well to read distance etc must be specified You start the run by cancelling the prerun and then clicking the Run button on the run sheet Once the Run button is clicked electrophoresis and data collection begin The raw data is stored in a gel file along with sample information from the run sheet imported from the sample sheet 60 80 100 120 140 160 180 Figure 1 1 Gel file Four different windows are available for viewing the data being collected and instrument status in real time The Scan and Gel windows are for viewing data The Status window displays the current status of the instrument The Electrophoresis History window displays the actual values for the electrophoresis power supply and gel temperature throughout the course of a run When the run is finished data is analyzed either automatically or manually Automatic data analysis must be specified on the run sheet prior to starting the run For more detailed information and instructions see Chapter 3 Instrument Operation and Chapter 9 Data Collection Software About This Instrument 1 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Data Analysis Overview Once the run is finished the collected data is transferred automatically if specified on the run sheet or manually to either the ABI PRISM DNA Sequencing Analysis Software or the ABI PRISM GeneScan Analysis Software for an
207. ere damage to the instrument Monitor the room temperature and humidity consult the following condensation tables and maintain the water bath temperature setting above the corresponding dew point Dew Point Table Ambient Temp 15 C Ambient Temp 20 C Ambient Temp 25 C Ambient Temp 30 C Ambient Temp 35 C Relative Approx Relative Approx Relative Approx Relative Approx Relative Approx Humidity Dew Point Humidity Dew Point Humidity Dew Point Humidity Dew Point Humidity Dew Point C C C C C 100 15 100 20 100 25 100 30 100 35 90 14 90 18 90 23 90 29 90 33 80 12 80 16 80 21 80 26 80 31 70 10 70 14 70 19 70 24 70 29 60 12 60 17 60 21 60 26 50 10 50 14 50 19 50 23 40 10 40 15 40 19 30 10 30 15 20 9 Subambient Temperature Operation B 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Parts and Accessories Appendix Contents In this Appendix Part numbers for the following are provided in this appendix Accessory Reagent See page Part Number Updates C 1 Glass Plates Spacers and Clamps C 2 Combs and Overlays C 3 Gel Cassette and Pouring Fixtures C 3 Buffer Chambers and Gasket Kits C 3 Electrophoresis Cable and Electrode Assemblies C 4 Front Heat Transfer Plate C 4 Two Pitch Eight Channe
208. erences 0 0 0 eee nerne 5 11 GeneScan Sample Sheet Preferences 0 0 0 eee cece eee ene 5 12 GeneScan Run Sheet Preferences 0 0 eee ete teen nee 5 13 General Settings er ciee e i eek eR Rah bare tes Gee A ia ented ao ink aie Tieden 5 15 Dye Indicator Preferences aa c c6 sie acd de satis Fae menika Rae ewok eee Sawa cena eo 5 17 Project Information Preferences 0 0 0 eee eee eee eee nee 5 20 6 Making Matrix Files for GeneScan 0 0 cece eee es Ol Chapter Contents 5i32 s4scnale nn We nga ea eee ed ape a gee Poked Uaa aa eia 6 1 Terminology staietseuteev a pede vega thee Gh td how es de ideee see edeedieoe hava 6 1 Purpose of the Matrix File for GeneScan Applications 0 0 00 e eee eee ee 6 2 Why Matrices Must Be Remade 0 0 0 00 cece eee eee nee 6 2 Evaluating Matrix File Quality 0 ce cee cette eee nee 6 3 Creating a Matrix File for GeneScan Applications 0 0 00 eee eee eee eee 6 6 Checking the Quality of a New Matrix File 0 0 ee cee eee 6 8 7 Making Instrument Files for Sequencing 7 1 Chapter Contents saarien ha ea ele aa tee arene E eee thaw baka ee ua are 7 1 TermimMolopgy sion peice oe iG cin is PAL E a dees ee bed ede 7 1 Instrument File Overview 0 0 eect eee ence tenn E 7 2 When to Make a New Instrument File 2 20 00 eee eee eee 7 3 The Data Utility Program cscs be sa eestit tiini esi Pee es CORSO be See eee ess
209. erences Action If the Preferences dialog box Then is not open open the Windows pull down menu select Preferences and then select GeneScan Sample Sheet Defaults is open open the Page pop up menu and select GeneScan Sample Sheet Defaults To change the size standard dye color open the size standard pop up menu and select the appropriate color B blue G green Y yellow R red Preferences Page GeneScan Sample Sheet Defaults w Size Standard Dye Color Cancel If Then you are finished click OK to save your changes or Cancel to quit without making any changes you wish to change more preferences open the Page pop up menu and select another Preference Preferences Step 1 2 3 5 12 Setting Preferences Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com GeneScan Run Sheet Preferences Setting GeneScan The following parameters can be set as preferences for GeneScan run sheets Run Sheet 4 Operator Preferences Well to read WTR distance in centimeters Lanes PreRun module Run module Autoanalyze Analysis Parameters Gel s Matrix File Size Standard Auto Print gt gt gt o o gt o o To change GeneScan run sheet preferences Step Action 1 If the Preferences dialog box Then is not op
210. ertion and removal much easier 3 6 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To load the gel into the cassette continued Step Action 7 Insert the teeth of the shark s tooth comb between the plates as follows a Hold the comb at both ends b Slowly and carefully insert the teeth of the comb between the plates without bending any of the teeth or introducing air bubbles c Continue sliding the comb between the plates until the tips of all the teeth penetrate the gel approximately 1 2 mm If the top of the gel is not completely flat in the sample loading region you may have to insert some of the teeth further than 1 2 mm so that all the teeth penetrate the gel surface IMPORTANT If any of the teeth penetrate the gel too much do not pull them out Leave them in the gel Removing teeth that have penetrated the gel causes samples to leak into adjacent wells 8 Proceed to Installing the Gel Cassette and Lower Buffer Chamber on page 3 8 Instrument Operation 3 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Installing the Gel Cassette and Lower Buffer Chamber The Electrophoresis The following illustration shows the various components of the electrophoresis Chamber chamber when it is empty Clamps High voltage connection Water flow for upper
211. es not yet exist leave the field set to lt none gt Required for IMPORTANT If the pop up menu lists lt none gt only software cannot find the folder that contains the instrument matrix files To correct this see Setting Folder Location Preferences on page 9 5 Selecting the Run To select the run mode well to read distance and operator Mode Well to Read Distance and Operator Step Action 1 Open the Well to Read distance pop up menu and select the appropriate value 2 If this is Then a standard instrument proceed to the next step an XL or 96 lane upgrade instrument verify that the correct mode is displayed in the Run Mode field Lanes 96 Lane Scan XL Scan Run Mode Full Scan 3 Click in the Operator field and type your name Data Collection Software 9 33 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Setting the To set the collection time Collection Time Step Action 1 Enter the duration of the run in the Collect time field Use the run time recommended in your protocol The following are suggested run times only Suggested Collection Time in Hours Long Ranger LR Type of Run PAGE PLUS PP Well to Read Scans per 19 1 Acrylamide 29 1 Acrylamide Length incm Hour Application Gels Gels 12 1200 GS 1 0 1 0 12 2400 GS 1 0 1 0 36 1200 GS am
212. et and sealant from the groove of the buffer chamber Be sure to remove all residual glue from the bottom of the groove IMPORTANT Do not use solvents to remove residual glue Solvents can damage the chamber 8 10 System Maintenance Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Installing the New Gasket To install the new gasket Step Action 1 Place the buffer chamber on clean lab paper to avoid scratching it 2 Wearing latex gloves apply a 1 16 1 5 mm diameter bead of sealant into the center of the groove Apply a consistent amount throughout the groove Place the gasket in the groove as follows a Hold the curves of the gasket as shown below and place the gasket into the groove b Gently press the gasket in place starting with both positions 1 the two curves of the gasket then position 2 and then each position 3 The gasket is longer than the groove Do not cut off the ends of the gasket otherwise the gasket will leak It is a sealed gasket that must remain sealed to work effectively GR1294 Remove excess sealant with a Kimwipe Be especially careful that no sealant is left on the surface of the gasket that will contact the glass plate Install the buffer chamber onto a cassette with glass plates for at least two hours before filling it with buffer This will ensure
213. f disorders the following recommendations have been developed to decrease awkward posture repetitive motion excessive force static muscle loading and soft tissue contact Use an automated multi channel pipette loader Locate the instrument on a variable or predetermined height worktable or lab bench Use a stable stool or stepladder Install adequate artificial lighting in the appropriate area to facilitate loading Ensure adequate front access to instrument while performing loading activities Remember to load the samples in the same lanes as specified on the run sheet The information on the run sheet at the start of the run is the information used to identify each sample in the Gel file Changes made to sample and run sheets after starting the run are not implemented To load the samples Step Action 1 If this is Then a 96 lane instrument proceed to Starting and Monitoring the Run on page 3 40 Sample loading instructions are part of this procedure not a 96 lane instrument If not already paused click Pause in the Run window to pause the PreRun Pausing stops electrophoresis but maintains the temperature of the gel while loading samples Instrument Operation 3 37 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To load the samples continued Step Action 2 Carefully flush all of the wells with 1X TBE
214. f ABI PRISM 377 Data Collection Software Improved and new accessories for the instrument New gel formulations and protocols User bulletins related to the use of this instrument will be mailed to you We recommend storing the bulletins in this manual A tab labeled User Bulletins has been included for this purpose About This Instrument 1 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Technical Support Contacting Technical You can contact Applied Biosystems for technical support by telephone or fax by Support e mail or through the Internet You can order Applied Biosystems user documents MSDSs certificates of analysis and other related documents 24 hours a day In addition you can download documents in PDF format from the Applied Biosystems Web site please see the section To Obtain Documents on Demand following the telephone information below To Contact Technical Contact technical support by e mail for help in the following product areas Support by E Mail Hours for Telephone Technical Support To Contact Technical Support by Telephone or Fax 1 6 About This Instrument Product Area E mail address Genetic Analysis DNA Sequencing galab appliedbiosystems com Sequence Detection Systems and PCR pcrlab appliedbiosystems com Protein Sequencing Peptide and DNA Synthesis corelab appliedbiosystems com Biochro
215. f the original installation and restart computer Error messages displayed during data collection See About Troubleshooting Software on page 4 20 for more information on possible causes Error messages displayed during data collection are also logged in the Log file The Log lists both computer errors and instrument firmware errors Computer cannot load a new firmware image onto the instrument or A new firmware image appears to have been downloaded however The instrument still does not operate The rear panel LEDs remain stuck in one pattern Instrument memory cleared due to a power outage Corrupted firmware on instrument Perform a total reset or cold boot the instrument See Performing a Total Reset on page 4 22 Performing a Cold Boot on page 4 23 4 10 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Signal Troubleshooting Guide Observation Possible Causes Recommended Actions Loss of signal 50 75 Plates and gel cassette not installed properly against positioning pins Repeat the run after reviewing the instructions in Chapter 2 for installing the plates and gel cassette Temporary loss of signal typically between 140 to 200 bp lasting about 20 to 40 bp Contaminants on glass plates See Temporary Loss of Signal on page 4 29 for more informa
216. fer plate and instrument sensitivity will be greatly reduced Over time gel solution can also damage the rear heat transfer plate 2 For square tooth combs only Place the overlay strip on the clean class Align the well outlines on the overlay strip with the teeth of the comb 3 Carefully remove the comb from between the plates and clean it with deionized water and Kimwipes Allow the comb to air dry 4 Clean the comb area with deionized water and Kimwipes Remove residual acrylamide from the well region Comb area Well region Very carefully use the teeth of the comb to remove residual acrylamide in the well region formed between the plates 5 Visually examine the plates for dust lint water spots and fingerprints Clean again if necessary 6 If the plates are Then mounted in the gel cassette proceed to Installing the Gel Cassette and Lower and you are using a Buffer Chamber on page 3 8 square tooth comb mounted in the gel cassette proceed to step 5 on page 3 6 and you are using a shark s tooth comb not in the gel cassette proceed to Loading the Gel Into the Cassette on page 3 6 Instrument Operation 3 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Loading the Gel Into the Cassette Procedure IMPORTANT When carrying a cassette with plates always hold the cassette by the sides with both hands Do not carry the cassette by the top bar The weigh
217. ference currently being set and corresponds to the select button at the bottom of the box 2 Use this pop up f Select Settings Folder menu to locate shee the desired folder fm ABI Folder 3 Click the folder C3 Acrobat Search Resources once only to Canecsott highlight it Co Apple Menu Items 7 AppleShare Folder Claris O Onen C3 Control Panels m Control Panels Disabled 4 Click here to set Select ABI Folder the preference Click the Select box underneath the list of folders step 4 in the illustration above Do not click Open If Then you are finished click OK to save your changes or Cancel to quit without making any changes you wish to change more preferences open the Page pop up menu and select another Preference Setting Preferences 5 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Default File Name Preferences Default File Name Data collection software automatically names the new files and folders created for Preferences each run These files and folders consist of Sample sheets Run folders Run files Gel files Sample files created after a run from the gel file gt gt gt Preferences Page File Names vj 8 Sample Sheet Sample Sheet lt date gt w Run Folder Run Folder 7A Run File BIA Sample File bE
218. fied on the run sheet 1 and3 Pause Temporarily halts the scanner No functions are performed The data in instrument memory continues to be stored and the gel temperature increases or is maintained as appropriate Resume Resumes instrument operation This command becomes active after instrument operation has been paused using the Pause command Cancel Run Active only when a module is running Allows you to immediately stop the run in progress The run is ended and the run sheet is closed A run cannot be resumed once it is cancelled 9 12 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Window Menu Window Status Log Sample Sheet Run Scan Gel Image Electrophoresis History The Window menu opens Electrophoresis History Manual Control window Preferences menu Manual Control Preferences Window menu commands The standard windows associated with a run Status Log Sample Sheet Run Sheet Scan Gel Image and Any other windows listed at the bottom of the menu Choosing any window from this menu brings that window to the front and makes it the active window For more Command Description detail see Status Displays the Status window page 9 53 Log Displays the Log file Command is active only when a page 9 54 module is running Sample Sheet Command active only
219. file name and the location where the sample sheet will be stored is displayed a Change the file name now if desired b Click Save IMPORTANT Although it is an option we do not recommend changing the storage location of the sample sheet If the location is changed software will not be able to locate the sample sheet when you set up the run sheet Folder where sample sheets are stored fg Sample Sheet 5 13 98 3 49 P fa Sample Sheet 5 13 fg Sample 1 7 Desktop fu Sample Sheet 5 fa Sample 5 15 98 3 Mf Save this document as Cancel f Sample sheet file name Note You can also save the sample sheet by opening the File menu and selecting Save Save As or Save A Copy In Data Collection Software 9 25 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com How to Enter Information on Sample Sheets Entering Sample Names Sample Info and Comments Moving From One Field to Another Applying Same Parameter to all Fields in a Column Copying Information Click in the Sample Name Sample Info GeneScan sample sheets only or Comments field Type the desired information More text can be entered in a field than is visible Text automatically shifts as it is entered Use the keyboard arrow keys to scroll through long entries Several methods can be used to move from one field to another Click in the field Press the directional
220. for a final concentration of 1X 1X TBE running buffer Instrument set up incorrectly Verify that The electrode leads are secure and plugged in The bottom and top of the gel are immersed in running buffer Broken instrument parts Inspect the electrophoresis cables electrodes and platinum wire in both buffer chambers If anything is visibly broken replace the electrophoresis cable and electrode assembly Procedure in Chapter 8 System Maintenance Corrupted firmware on instrument Resend firmware by performing a total reset See Performing a Total Reset on page 4 22 Front heat transfer plate sticks to the gel plates Upper buffer chamber leak Follow this procedure a Empty and remove the upper buffer chamber Open the plate clamps c Slide the front heat transfer plate up along the gel plate until it is released d Clean the front heat transfer plate e Check the upper buffer chamber for leaks and repair or replace as appropriate IMPORTANT Always remove the front heat transfer plate from the gel cassette before removing the cassette from the instrument The front heat transfer plate is heavy Removing them together can damage the cassette Troubleshooting 4 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Data Collection Software Troubleshooting Guide Observation Possibl
221. for analyzed data to be printed automatically Proceed to Starting the Run or Closing the Run Sheet on page 3 34 3 32 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Auto Analysis for See About Automatic Data Analysis in Chapter 9 for more information GeneScan Runs IMPORTANT Do not analyze matrix standard samples To set up the run sheet for automatic data analysis Step Action 1 Verify that the data analysis software is selected in Preferences To do this a Open the Window menu and select Preferences b Select GeneScan Run Defaults c Look at the Autoanalyze with field Preferences SSS Page GeneScan Run Defaults Y Operator wIR 12 w cm Lanes 24 v PreRun Matule Run Mue C emoe F s Analysis software Bi Autoanalyze with Analysis Parameters Analysis Default v not selected oatsretricfe Size Standard Auto Print Co If the analysis software is Then selected click OK to return to the run sheet not selected a Open the Autoanalyze with pop up menu and select Other b In the dialog box locate the ABI PRISM GeneScan Analysis Software and click Open c Close the current run sheet and create a new run sheet Note Changing the preference has no affect on run sheets cre
222. for answers to frequently asked Support Through questions and for more information about our products You can also order technical the Internet documents or an index of available documents and have them faxed or e mailed to To submit technical questions from North America or Europe you through our site The Applied Biosystems Web site address is http Avww appliedbiosystems com techsupp Step Action 1 Access the Applied Biosystems Technical Support Web site 2 Under the Troubleshooting heading click Support Request Forms then select the relevant support region for the product area of interest 3 Enter the requested information and your question in the displayed form then click Ask Us RIGHT NOW blue button with yellow text 4 Enter the required information in the next form if you have not already done so within 24 to 48 hours then click Ask Us RIGHT NOW You will receive an e mail reply to your question from one of our technical experts To Obtain Free 24 hour access to Applied Biosystems technical documents including MSDSs Documents on Demand is available by fax or e mail or by download from our Web site To order documents Then by index number a Access the Applied Biosystems Technical Support Web site at http Avww appliedbiosystems com techsupp b Click the Index link for the document type you want then find the document you want and record the index number
223. front of tab the lid rests on tab is located of the buffer chamber on the rear of the buffer chamber Transparent hale buffer chamber J White buffer chamber Wait approximately 30 seconds and then check for leaks as follows a Look for fluid dripping down the front of the plates This indicates the buffer chamber gasket may be worn and should be replaced b Look for fluid inside the lower buffer chamber This indicates that buffer is leaking over the ears of the plates and down the sides of the cassette 3 16 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To fill the buffer chambers Step Action 4 If leakage occurs try one or more of the following Check the buffer level If the chamber is overfilled remove the excess buffer Plug the suspected areas with melted agarose If buffer is leaking over the ears of the plates apply a bead or ribbon of agarose to seal this area Carefully empty the buffer chamber and clean the gasket Also clean the front of the glass plate where the gasket makes contact If leakage continues replace the gasket Follow the procedure Replacing the Upper Buffer Chamber Gasket in Chapter 8 IMPORTANT If the buffer level drops below the notch in the front plate in the upper buffer chamber or below the bottom of
224. ftware program disks or copies of the programs are located should it become necessary to reload all the system software See Backing Up Important Files on page 8 20 and System Software on page 8 25 System Maintenance 8 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Backing Up Important Files Overview Backing up means making a copy of the programs and files present on the hard disk and storing the copies in a location other than the hard disk of the computer that runs the instrument Various methods can be used to backup files such as gt gt Copying the files onto a portable disk such as a Zip disk Copying the files to a server on a network Copying the files onto a hard drive other than the one used to run the instrument Using Disk Copy to make image files of software programs from the original floppy disks directly onto a hard drive or other storage media Recommendations To avoid losing data files and software programs in the event of a hard disk failure we strongly recommend 8 20 System Maintenance Backing up all important data files gel files sample files run sheets and sample sheets once each day Making a copy of each important software program using an application such as Disk Copy Backing up other important files the same day they are created e g custom modules matrix instrument files size standard files analysis para
225. ftware programs ABI PRISM GeneScan Analysis Software ABI PRISM DNA Sequencing Analysis Software The following information is a brief overview of the data analysis process up through the generation of sample files For more information on these software programs and how to use them to analyze your data refer to the following publications ABI PRISM DNA Sequencing Analysis Software User s Manual ABI PRISM GeneScan Analysis Software User s Manual Sample Files When a run is finished the gel file is processed by sequencing or GeneScan analysis software either automatically or manually The Auto Analyze feature and associated parameters on the run sheet must be properly configured for data analysis to occur automatically at the end of a run See About Run Sheets on page 9 28 for more information Gel file processing includes Examining and adjusting the gel image Adjusting the tracking and marking lanes for extraction Extracting sample data for each lane marked and generating sample files One sample file is created for each lane of data extracted from the gel file Each sample file contains Information entered on the sample and run sheets Raw and analyzed data Run start and stop times Voltage temperature and power values during the run A record of analysis settings Peak locations and size calling values gt gt gt o ad Base calls for sequencing applications only Sample files are stored in the
226. ge notch in the front plate slowly and steadily inject gel solution between the plates across the entire width of the notch t 4t ii Bi i EO GR1103 yop 4 Simultaneously Continue injecting the gel solution while moving the syringe back and forth along the width of the notch Constantly tap the top plate directly in front of the gel path to eliminate air bubble formation Injection takes approximately 60 seconds Stop when the solution completely fills the space between the plates Note If you need to refill the syringe be careful not to introduce any air bubbles Keep excess solution in the beaker to verify polymerization 2 22 Pouring Gels Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To inject the gel solution continued Step Action 5 If using a Then shark s tooth comb insert the straight edge of a shark s tooth comb to form one large well square tooth comb insert the teeth of a square tooth comb to form the wells 6 Verify no air bubbles are trapped where the gel and comb meet Attach three large binder clips to the top of the plates directly over the comb Evenly space the clips If using a shark s tooth comb be careful not to damage any teeth while clamping the plates IMPORTANT Do not attach binder clips to the bottom of the gel This can result
227. gel for single strand conformation polymorphism SSCP Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Importance of Using High Quality Gels Why a High Quality One of the most critical variables that determines the success or failure of both Gel is Important sequencing and GeneScan runs is the gel The use of consistently prepared high quality gels helps ensure the best experimental results and minimizes the time spent troubleshooting gel related problems Poor quality gels often cause problems that are mistaken for instrument problems For sequencing runs the quality of the gel directly effects the number of bases that can be called For GeneScan runs the quality of the gel effects the mobility of DNA fragments from run to run reproducibility of sizing signal strength and resolution Factors that affect gel quality include Purity and freshness of reagents Rate of polymerization Presence of air bubbles Age of the gel A discussion of these factors is located in Appendix A Gel Recipes Pouring Gels 2 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com About the Gel Pouring Methods in This Chapter Methods Presented in This Chapter Method 1 Method 2 Summary of the Gel Preparation Procedure 2 4 Pouring Gels Many methods and devices are available for pouring gels The two methods pre
228. good adhesion and allow the sealant to cure System Maintenance 8 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Replacing Electrophoresis Cables and Electrodes Overview Electrophoresis cables and electrodes are built as assemblies to simplify their installation Therefore change the entire assembly when replacing a cable or electrode Part numbers for the assemblies are as follows Buffer Chamber Electrode Assembly Part Number Upper 254304 Lower 254303 Procedure ELECTRICAL SHOCK HAZARD The ABI Prism 377 contains a high voltage power supply Although the instrument has been designed with safety features in the door to disconnect the power supply when the door is open please follow procedures as prescribed As with any electrophoresis apparatus be careful during instrument operation and when handling electrodes and liquids To replace an electrophoresis cable and electrode assembly Step Action 1 Disconnect the electrophoresis cable from the instrument and remove the buffer chamber 2 To remove the cable electrode Follow these steps assembly from the upper buffer chamber a Remove the two plastic screws on the back left end of the upper buffer chamber Figure 8 1 or Figure 8 2 on page 8 13 b Remove the assembly from the buffer chamber lower buffer chamber The assembly in the lower buffer chamber is held und
229. h and time during degassing A 7 reset single reset performing 4 22 total reset performing 4 22 resolution affects of acrylamide impurity A 5 definition of 4 34 deterioration due to aging gel A 8 modifying run voltage effect of 4 34 to 4 35 troubleshooting guide 4 17 to 4 18 restarting the computer run cleaning up after run 3 45 commands to perform 9 39 to 9 41 description of 1 17 modifying run time 4 34 9 45 starting and monitoring 3 40 to 3 43 run file saving 8 23 run sheets for GeneScan runs 9 29 to 9 30 for sequencing runs 9 28 to 9 29 how to prepare 3 28 to 3 34 9 31 to 9 37 preferences setting GeneScan 5 13 to 5 14 sequencing 5 11 to 5 12 viewing 9 57 run temperature determining 3 36 9 43 modifying 9 45 viewing in Status window 3 36 run time modifying 4 34 9 45 run window Same as run sheet runs folder location and description of 3 51 8 18 S safety information ergonomic hazards and recommendations 1 4 Site Preparation and Safety Guide 1 4 safety information location 1 3 safety warnings electrical shock hazard 1 4 4 32 8 2 8 7 8 10 8 12 general warnings for ABI Prism 377 1 4 TEMED A 6 A 23 urea A 6 SAM Symantec AntiVirus program about 8 21 sample files archiving 8 23 9 58 description of 9 60 location of 3 51 9 4 making instrument file 7 18 to 7 19 sample loaders two pitch eight channel loader suppliers C 5 to C 6 sample loading suggested load volumes 3 39 sample mobility
230. harmful by inhalation contact with the skin and if swallowed Use only in a chemical fume hood When handling wear lab coat safety glasses and chemical resistant gloves Immediately pour the gel Allow the gel to polymerize for at least 2 hours before use A 20 Gel Recipes Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com For SSCP To prepare a standard 6 5 nondenaturing polyacrylamide gel for SSCP Applications Only Step Action 1 Instructions for preparing glass plates and two gel pouring methods are located in Chapter 2 Pouring Gels Glass plates and all other gel pouring equipment must be ready for use prior to adding the polymerizing reagents to the gel solution Combine the following 6 5 mL 40 polyacrylamide stock 37 5 1 1 0 g mixed bed ion exchange resin 1 25 g glycerol 25 mL distilled deionized water Mix well to obtain a homogenous solution Note This step is critical because glycerol is viscous and it is difficult to obtain a homogeneous solution Use a 0 2 um cellulose nitrate filter to filter the solution for 5 minutes Note During this step you both filter and degas the solution Add 4 0 mL of filtered 10X TBE and sufficient distilled deionized water to bring the total volume to 40 0 mL IMPORTANT If the plates are not clean and mounted in the gel cassette or other device c
231. he information imported from the sample sheet cannot be changed on the run sheet If changes are made to the sample sheet after it has been selected on the run sheet you must reimport the sample sheet data To make changes to the sample sheet after selecting it on the run sheet a Open the sample sheet by clicking the icon next to the sample sheet pop up menu on the run sheet Sample Sheet Sample Sheet 5 1 v Click this icon Make changes to the sample sheet Close and save the sample sheet Open the sample sheet pop up menu on the run sheet and select lt none gt oaos Open the sample sheet pop up menu on the run sheet and reselect the sample sheet Note You cannot make changes to the sample sheet while a module is running Select the For GeneScan runs in particular matrices are dye set instrument and run condition Instrument or Gel s dependent As such matrices must be remade when any of these conditions change Matrix File For more information refer to Chapter 6 Making Matrix Files for GeneScan and the ABI Prism GeneScan Reference Guide P N 4303188 To select an instrument or gel matrix file If thisisa Then sequencing run open the Instrument File pop up menu and select the appropriate file If one does not yet exist leave the field set to lt none gt A file must be selected for automatic data analysis GeneScan run open the Gel s Matrix File pop up menu and s
232. he load volume or b Close the front panel of the instrument to Suggested Load Volumes on page 3 39 c Click Resume and allow the samples to electrophoresis into the gel for two minutes Click Pause Open the front panel of the instrument f Carefully flush all of the wells with 1X TBE buffer to remove any residual formamide from the previously loaded wells Change the pipet tip for each sample g Load one sample into each even numbered well IMPORTANT Whenever possible load a sample in lane one unless contaminants are present and you are skipping that lane It is better to leave empty lanes at the right end of the gel Also avoid skipping more than one lane between samples The tracker may not work properly if more than one lane is left empty between samples IMPORTANT For shark s tooth comb users loading samples in every other lane is necessary for good tracking Although the formamide in the loading solution helps narrow the lane width loading samples in adjacent lanes can blur the definition between lanes Loading samples in every other lane creates discrete spaces between samples to properly identify the lanes Close the front panel of the instrument and proceed to Starting and Monitoring the Run on page 3 40 3 38 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Suggested Load Total resuspensio
233. he peak apex and the W are the peak widths measured at half peak maximum Determining Required Run Time To determine the minimal acceptable run time for a given run voltage you will need to perform a trial run To ensure that you collect sufficient data to perform analysis set the electrophoresis run time approximately 10 higher than the migration time of the largest fragment of interest In general the migration time of the 400 bp fragment in the GeneScan 400HD Internal Lane Size Standard is 2 hours on a 36 cm well to read 5 Long Ranger gel Note The largest fragment of interest will most probably be a size standard peak that is needed for sizing the largest sample fragments of interest The set of size standard peaks that GeneScan uses to generate the sizing curve can vary with the size calling method In general be sure to include the two size standard peaks immediately larger than the largest sample fragment of interest Decreasing Run Time For faster run times you can increase the electrophoresis voltage but this can decrease the resolution Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Modifying Perform native applications and non denaturing applications such as SSCP at lower Electrophoresis temperatures 27 42 C Temperature Protocols for most denaturing applications specify a 51 C run temperature For More For information on setting electrophoresis
234. he white upper buffer chamber The version released in mid 1998 illustrated on page 8 10 P N 4304406 is transparent with a flat window and is referred to in this section as the transparent upper buffer chamber Separate instructions are provided for each chamber For the Transparent Upper Buffer Chamber Parts and Tools Kit Part Number Supplier must fit into groove of buffer chamber to remove old sealant ABI PRISM 377 Buffer Chamber Gasket 7307172 Applied Biosystems Kit contains 2 gaskets P N 430440 sealant P N 201480 and instructions Latex gloves N A Major laboratory supplier Kimwipes or similar lint free wipes N A Major laboratory supplier Spatula N A Major laboratory supplier For the White Upper Buffer Chamber Parts and Tools Kit Part Number Supplier must fit into groove of buffer chamber to remove old sealant ABI PRISM 377 Buffer Chamber Gasket 604524 Applied Biosystems Kit contains 5 gaskets sealant and instructions Latex gloves N A Major laboratory supplier Kimwipes or similar lint free wipes N A Major laboratory supplier Scrap glass plate or flat rigid plastic plate N A Major laboratory supplier at least 10 x 4 Spatula N A Major laboratory supplier Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Procedure for White The following is an illustration of the white
235. hed Instructions are listed under Auto Analysis for Sequencing Runs on page 9 35 To perform automatic data analysis after a GeneScan run the following information must be selected on GeneScan sample and run sheets Onthe GeneScan sample sheet Size standard designation Std column Dye colors run in each lane Pres column Onthe GeneScan run sheet Gels matrix file Matrix file Analysis parameters Size standard Auto analyze Auto print optional In addition the analysis program ABI PRISM GeneScan Analysis Software must be selected on the GeneScan Run Defaults Preference This tells the data collection software where to send the data when the run is finished Instructions are listed under Auto Analysis for GeneScan Runs on page 9 36 9 38 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Commands Used to Perform a Plate Check PreRun and Run Overview A plate check prerun and run can be started by using the buttons on the run sheet or by using commands from the Instrument menu Using the Buttons A button must be active to be selected on the Run Sheet Buttons looks like this when active Toggles between Pause and Resume Buttons looks like I Psese E cansi this when inactive The following is a description of each button and its function Button Function
236. hemistries 7 4 terminology 7 1 verifying instrument files 7 16 to 7 17 when to make new file 7 3 run sheets about 9 28 to 9 29 preparing 9 31 to 9 37 setting preferences 5 11 to 5 12 sample sheets about 9 14 to 9 15 entering information 9 26 importing exporting information 9 27 preparing 9 16 to 9 19 setting preferences 5 10 serial number entering in Calibration file 4 27 8 15 settings ABI folder location and description of 3 52 settings changing manually 3 48 shark s tooth comb inserting 3 6 signal description of temporary loss 4 29 Index 8 preventing temporary loss of 4 29 troubleshooting guide 4 11 to 4 15 single reset 4 22 Single stranded Conformation Polymorphism See SSCP Site Preparation and Safety Guide description of guide 1 4 skipping lanes determining which to skip 3 14 software adding non Applied Biosystems software 8 28 additional ABI Prism software AutoAssembler 1 21 to 1 22 BioLIMS 1 22 GenBase 1 20 GeneAssist Sequence Analysis System GenoPedigree 1 20 Genotyper 1 20 Primer Express 1 20 Sequence Navigator chiller modules description of 9 44 data analysis software described 1 18 to 1 19 Data Collection software files and folders 9 3 to 9 4 menucommands 9 9 to 9 13 starting 9 8 DataUtility program about 1 19 description of program 7 3 using to verify instrument files 7 16 to 7 17 downloading from the web 8 26 to 8 27 GelDoc II utility 1 19 modules 9 42 to 9 48 chiller modules inst
237. hoose Update File instead of New File Select the instrument file to be modified b For step 9 click Open instead of Save 13 Quit the DataUtility program 14 Verify the instrument file by following the procedure Checking Instrument Files on page 7 16 Making Instrument Files for Sequencing 7 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Making an Instrument File for Virtual Filter Set E from Matrix Standards Overview When creating an instrument file for virtual filter set E you must create three matrix files dye primers Taq terminator and T7 terminator All three matrix files must be made even if you are only using one dRhodamine based chemistry Data collection software will not run with only a Taq or T7 terminator matrix in the file The T7 Terminator Matrix file is needed to analyze dRhodamine terminator and BigDye terminator sequencing data It has a baselining algorithm associated with it that works well with these chemistries The dRhodamine terminator and BigDye terminator dye set primer files have tags in them that cause the Sequencing Analysis software to select this matrix file The correct placement of standards in the data utility application for making a Filter Set E instrument file is shown in the table below Table 7 1 Placement of Standards for Virtual Filter Set E Dye Primer T7 Terminator
238. ic Areas Served World Precision Liegnitzer Str 15 Austria ete Inc D 10999 Berlin Germany Bulgaria Voice 49 0 30 6188845 Czechoslovakia Fax 49 0 30 6188670 E mail wpi wpi sireco de Germany Greece Holland Netherlands Hungary Italy Poland Rumania Russia Switzerland Yugoslavia World Precision 1 4 2 702 Naka Meguro Meguro Japan Instruments Inc Tokyo 153 0061 Japan Japan Voice 81 0 3 3760 5050 Fax 81 0 3 3760 5055 E mail wpi tkb att ne jp World Precision Astonbury Farm Business Centre Belgium Instruments Inc Aston Stevenage i D k United Kingdom Hertfordshire SG2 7EG England SA England Voice 44 0 1438 880025 l Fax 44 0 1438 880026 Finland E mail wpi piuk demon co uk France Ireland Norway Portugal Scotland Spain Sweden World Precision Sarasota International Trade Center Areas not listed above Instruments Inc Other world wide areas 175 Sarasota Center Blvd Sarasota FL 34240 9258 USA Voice 941 371 1003 Fax 941 377 5428 E mail wpi wpiinc com C 6 Parts and Accessories Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com User s Manuals Instrument 4303613 ABI PRISM 377 DNA Sequencer User s Manual 904412 ABI PRISM 377 DNA Sequencer XL Upgrade User s Manual 4305423 ABI PRISM 377 DNA Sequencer 96 lane Upgrade
239. ies ABI AutoAssembler BigDye Factura GenBase GeneAssist GenoPedigree and Primer Express are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries AmpliTaq GeneAmp and TaqMan are registered trademarks and AmpliTaq Gold is a trademark of Roche Molecular Systems Inc Long Ranger is a trademark of The FMC Corporation Macintosh is a registered trademark of Apple Computer Inc All other trademarks are the sole property of their respective owners P N 4307164B Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Contents 1 About This Instrument 0 ccc cece ee eee ee eee ee del Chapter Contents susiy psa oe deena ad Meet dad A ee eae Arte Se deed eae 1 1 Getting Started Quickly cis 0 nace ceca e qe dea ee daae ad ene see ee ee eae SNE See wees wales 1 2 What to Do If You Are a New User 1 cence eens 1 3 Safety Information 4 i462 das ed eritu SEEE EERE EEE SEE KEES PEER EE Rea aa S 1 4 Special Text Usage ci discs arie sunig Pas E EEN E viadaad aha d age edie T Peed di Ane g E ia 1 5 User Bulletins sc cig iat wane iea ade eee ake bad wey Asie ale ee eee 1 5 Technical Suppott ccc eee aan naa epee eee pana ee ee ode Seas we eae he 1 6 ABI PRISM 377 DNA Sequencer System Components 00 00 e eee eee 1 11 Theory of Operations vei ddrt ostiak sede a E E Mea EERE weds 1 13 Using the Instrument ricit chew bene e
240. if a sample sheet is open in the page 9 14 background while a module is running Sample and 9 20 information can be changed However the changes will not affect the run in progress Run Command active only if a run sheet is open in the page 9 28 background while a module is running Information cannot be changed once the Run button is clicked Scan Displays the Scan window Command is active only page 9 55 when a module is running Gel Image Displays the Gel File Command is active only when a page 9 55 module is running Electrophoresis Displays the Electrophoresis History window Command page 9 56 History is active only when a module is running Manual Control Displays a window that allows you to manually control page 9 57 certain instrument functions Preferences Opens the Preferences menu Chapter 5 Setting Preferences Data Collection Software 9 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com About Sample Sheets for Sequencing Applications What isa A sequencing sample sheet shown below is a file that contains information used for Sequencing Sample identification and tracking Sample Sheet Data analysis There are two types of sample sheets one for sequencing and one for GeneScan analysis applications The information entered on the sample sheet is imported to the sequencing run sheet described on page 9 28 IMPORTANT Do not mix seq
241. ile menu and select New leave the run window open 2 Click the icon named GeneScan Sample 3 Enter sample names in the Sample Name column by clicking in the appropriate Sample Name field and typing the sample name Enter names in the exact order the samples will be loaded onto the gel The numbers to the left of the Sample Name column represent the gel lane numbers Leave fields blank that correspond to empty lanes See Figure 3 4 on page 3 25 IMPORTANT Each sample must have a unique name Limit sample names to 27 characters including the default characters Do not use colons slashes or symbols in sample names Note More text can be entered than is visible Text automatically shifts as the information is entered Use the keyboard arrow keys to scroll through long entries To import sample names from tab delimited text files follow the instructions listed under Importing and Exporting Sample Sheet Information in Chapter 9 4 If matrix standard samples are being run enter a name for each matrix sample To help ensure a robust matrix is produced we strongly recommend you follow these guidelines see Figure 3 4 on page 3 25 Leave at least one empty lane between non matrix standard samples and matrix standard samples Leave one empty lane between each matrix standard sample IMPORTANT Matrices are dye set instrument and run condition dependent As such matrices must be remade when any of these conditions change For mor
242. ile menu and select Quit 7 Quit the internet connection and continue the installation by following the instructions listed under Loading from Floppy Disk to Hard Drive below Loading from To load the software from the floppy disk onto the hard drive Floppy Disk to Hard Drive Step Action 1 Quit any currently running applications 2 Hold down the Shift key and choose Restart from the Special menu to restart the computer with extensions turned off Continue holding down the shift key until you see the extensions are all disabled IMPORTANT If extensions are not turned off you may not be able to complete the installation 3 Insert the disk with the software into the floppy drive of the Macintosh connected to the instrument If the Installer window does not open automatically double click the disk icon Open the Readme file and read it Double click the Installer icon Click Quit when installation is complete 4 5 6 7 Respond to the prompts by clicking Continue or Install 8 9 Close the installer window and drag the disk icon to the trash to eject the disk 10 Store the disk for future use 11 Restart the Macintosh to turn the extensions back on and rebuild the desktop See Rebuilding the Desktop on page 8 18 12 Launch the new software If the old Preferences file was renamed as recommended under Renaming the Existing Software and Preferences on page
243. ilter set combinations The display colors represent the relative not the actual detection wavelengths For consistency the software always displays analyzed data with A as green C as blue G as black and T as red in the electropherogram view The wavelengths of the windows in the virtual filter sets used in cycle sequencing applications are listed in the following table Wavelength Range of Virtual Filter Virtual Filter Set Color nm A blue 530 541 green 554 564 yellow black 581 591 red 610 620 E blue 535 545 green 565 575 yellow black 590 600 red 620 630 Data Collection Software 9 51 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Filter Sets Available All the ABI PRISM 377 instruments use six virtual filter sets designated A through F For GeneScan applications Virtual filter sets A C D and F are used For Sequencing Applications Virtual filter sets A and E are used For More For more information see Information About Run Sheets on page 9 28 Modules on page 9 42 GeneScan Reference Guide Automated DNA Sequencing Chemistry Guide 9 52 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Viewing Data and Instrument Status Windows Available Four different windows are available for viewing real time data and instr
244. in an uneven gel thickness IMPORTANT To prevent well leakage the 96 lane plates and casting combs require 10 12 pounds clamping pressure 5 Do not put clips on bottom of gel 8 Cover the bottom of the plates with plastic wrap or damp paper towel to keep the bottom of the gel from drying out 9 Allow the gel to polymerize the amount of time recommended by the gel solution protocol Pouring Gels 2 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Index C chemistries gels used for GeneScan runs 3 2 gels used for sequencing runs 3 2 combs cleaning 3 8 D dishwasher recommendations 3 8 using to clean plates 3 6 G gasket mark description of 3 5 gel cassette how to carry with plates 3 10 gel injection device fixture 3 14 gel pouring methods overview of methods 3 4 plates mounted in cassette 3 10 using unmounted plates 3 20 gel quality factors that affect 3 1 importance of 3 3 gel recipes See Appendix A 3 1 gel recommendations 3 2 gel sapcers See spacers gels selecting a gel formulation 3 2 using high quality gels importance of 3 3 using new glass plates 3 5 See Also gel recipes glass plates avoid organic solvent 3 7 before using new plates 3 5 cleaning by hand 3 6 to 3 7 cleaning in a dishwasher 3 6 gasket mark 3 5 identifying front and back 3
245. in the analyzed data Dye set primer files also tell the software which matrix in the instrument file to use for data analysis Dye set primer file names indicate the primer chemistry type and concentration of gel and sometimes the filter set used The abbreviations used for these file names are as follows Abbreviation Definition DP Dye primer DT Dye terminator X The approximate percent concentration of the gel used Ac Acrylamide gel LR Long Ranger gel BD BigDye dR dRhodamine x x x Any combination of virtual filter set primer and chemistry Note The percentages 4 5 and 6 are approximates For example if the gel is 4 25 acrylamide use the corresponding 4 acrylamide dye set primer file Also 5 Long Ranger equals 4 acrylamide when selecting these files The following examples show how to interpret a dye set primer file name DyeSet Primer File Name Definition DP4 Ac 21M13 DP dye primer 4 Ac 4 acrylamide gel 21M13 the primer DP5 LR BD M13 FWD amp REV DP dye primer 5 LR 5 Long Ranger gel BD Big Dye M13 FWD amp REV m13 forward and reverse primers Data Collection Software 9 49 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com DyeSet Primer File Name Definition DT dR Set Any Primer DT dye terminator dR dRhodamine dR
246. ine Terminator Cycle Sequencing Kits or the dRhodamine Matrix Standards Kit only Use virtual filter set D and F modules with GeneScan and NED dyes 2 These chiller modules are recommended for PCR Single stranded Conformation Polymorphism Analysis SSCP Data Collection Software 9 47 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com ABI PRISM 377 Data Collection Software version 2 1 module files continued GS PR 36A 2400 GS PR 36A 2400 CHILLER GS PR 36D 2400 GS PR 36D 2400 CHILLER GS PR 36C 2400 GS PR 36C 2400 CHILLER GS PR 36F 2400 GS PR 36F 2400 CHILLER GS Run 12A 1200 GS Run 12A 1200 CHILLER GS Run 12C 1200 GS Run 12C 1200 CHILLER GS Run 12D 1200 GS Run 12D 1200 CHILLER GS Run 12F 1200 GS Run 12F 1200 CHILLER GS Run 12A 2400 GS Run 12A 2400 CHILLER GS Run 12C 2400 GS Run 12C 2400 CHILLER GS Run 12D 2400 GS Run 12D 2400 CHILLER GS Run 12F 2400 GS Run 12F 2400 CHILLER GS Run 36A 1200 GS Run 36A 1200 CHILLER GS Run 36C 1200 GS Run 36C 1200 CHILLER GS Run 36D 1200 GS Run 36D 1200 CHILLER GS Run 36F 1200 GS Run 36F 1200 CHILLER GS Run 36A 2400 GS Run 36A 2400 CHILLER GS Run 36C 2400 GS Run 36C 2400 CHILLER GS Run 36D 2400 GS Run 36D 2400 CHILLER GS Run 36F 2400 GS Run 36F 2400 CHILLER GS Run 60W A CHILLER GS Run 6
247. ing 3 13 laser power function description of 3 49 laser run function description of laser standby function description of 3 49 load volumes suggested 3 39 loading samples how to 3 35 skipping lanes 3 38 loading solution preparing loading solution 3 3 using formamide in empty wells 3 38 3 49 Log file description of 9 54 description of and viewing 3 44 errormessages 4 20 Log window See Log file Long Ranger gels 10 ammonium persulfate A 15 4 75 Long Ranger Singel A 16 5 Long Ranger gel for SSCP A 17 not for SSCP A 16 to A 17 gel solution ingredients A 15 recommendations A 2 run conditions for 48 and 36 cm gels A 15 lower buffer chamber filling 3 16 installing 3 8 to 3 10 M Macintosh computer data flow to instrument maintenance adding non Applied Biosystems software 8 28 archiving data from runs 8 23 to 8 24 backing up files 8 20 deleting data files 8 18 to 8 19 downloading software from the web 8 26 to 8 27 optimizing hard disk 8 19 rebuilding desktop 8 18 recommendations 8 17 reinstalling software 8 25 restarting computer 8 18 system file about 8 29 virus prevention program 8 21 Norton Utilities 8 22 operating error messages 4 20 Symantec Antivirus program SAM 8 21 maintenance computer See Macintosh computer maintenance instrument CCD pixel position value 8 14 to 8 16 cleaning accessories 8 3 to 8 4 recommendations 8 2 refilling water reservoir 8 5 replacing electrophoresis cables and electr
248. ing the Buffer Chambers on page 3 16 Instrument Operation 3 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Filling the Buffer Chambers To Fill the Buffer IMPORTANT The concentration of the buffer in both chambers must match the Chambers concentration of the buffer in the gel Otherwise samples will run slower or faster than expected or you may get a No EP Current Detected error message The gel recipes listed in Appendix A use 1X Tris Borate EDTA buffer TBE WARNING Tris borate EDTA TBE buffer can be harmful if inhaled ingested or absorbed through the skin It is irritating to the eyes skin and mucous membranes Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves To fill the buffer chambers Step Action 1 If not already prepared prepare the 1X Tris Borate EDTA TBE buffer solution by following the instructions listed on page 3 3 Slowly and carefully fill the upper buffer chamber with approximately 600 mL of 1X TBE buffer Do not allow buffer to splash onto the plates Fill levels for the two styles of upper buffer chambers for this instrument are shown below Filling the buffer chamber to the recommended level helps reduce the amount of buffer that evaporates during a run Fill to top of the two level marks Fill so buffer just touches the bottom located on each side of the
249. int dialog box depends on the printer you are using 4 Click Print Note If you choose Auto Analyze and Auto Print on the run sheet electropherograms are automatically printed after the collected data is analyzed 9 58 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Quitting the Data Collection Program Closing Open To close open windows Windows Step Action 1 Click a window to make it active 2 Close the window using one of the following methods Click the close box in the upper left corner of the window Open the File menu and choose Close The current settings in the window are saved Quitting the To quit the data collection program Program Step Action 1 Open the File menu and choose Quit Note Ifa run is in progress a dialog box asks you to verify that you want to quit Quitting the data collection software program while a run is in progress cancels the run Whatever data has been collected to that point is saved You can then use either analysis program to open the Gel file containing the collected data Data Collection Software 9 59 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Data Analysis Overview Overview When the run is finished data is analyzed automatically or manually using one of two data analysis so
250. int fields Auto analyze selected A set of analysis parameters and a size standard must be selected Run Sheet Example Run Module 65 Run 56A 24 Sample Sheet SampleSheetExa w Gel s Matrix File A Primer Matrix v a Figure 3 6 Auto analyze selected and deselected on a GeneScan run sheet Starting the Run or To start the run or close the run sheet Closing the Run Sheet If Then you are ready to proceed a open the File menu and choose Save A dialog box with instrument operation showing the default run sheet name and the location where the file will be saved is displayed Change the file name and storage location now if desired Folder where run sheets are stored i Run Folder 5 17 98 1 06 Y Macintosh HD m Desktop ew Save this document as Cancel Run sheet file name Run 5 17 98 1 15 PM L Click Save Proceed to Performing a PreRun and Loading the Samples on page 3 35 you wish to close the run sheet open the File menu and choose Close Click Save A dialog box showing the default run sheet name and the location where the file will be saved is displayed shown in previous step Change the file name and storage location now if desired Click Save 3 34 Instrument Operation Artisan Technology Group Qual
251. ion of the CCD Pixel Position Value and Checking and Entering the CCD Pixel Position Value on page 4 26 Creating the ABI 377 Calibrations File To create an ABI 377 Calibrations file Step Action 1 Turn power on to the instrument 2 Select Manual Control from the Window menu 3 Open the Fxn Name pop up menu 4 Select Calibration File Make 5 Click Execute and select Save from the File menu Adding the Serial Number to the ABI 377 Calibrations File Add the instrument serial number to the ABI 377 Calibrations File To enter the instrument serial number in the calibrations file Step Action 1 Make a note of the serial number from the back of the instrument 2 Open the System folder then the Preferences folder and then the ABI 377 Calibration file 3 Select the series of question marks displayed and then type the instrument serial number in their place es SS ABI 377 Calibrations E 00206 CCD X Skip Open the File menu and select Save Close the ABI 377 Calibrations file 6 Quit the SimpleText program Troubleshooting 4 27 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Using Calibration To send the ABI 377 Calibrations file information to the instrument File Send Step Action 1 Turn power on to the instrument Open the Window menu and select Manual Contro
252. ion software Set the folder locations preferences See Folder Location Preferences on page 5 5 for more detailed instructions Reset the remaining preferences that you use Detailed instructions are listed by preference in this chapter 5 4 Setting Preferences Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Folder Location Preferences Setting Folder The folder locations for the following types of files and folders must be designated to Location Preferences perform a run and to handle the data generated during a run Sample sheets Module files Run folders Firmware image file Settings GeneScan analysis parameters for GeneScan users only 5 gt gt gt a gt o GeneScan size standards for GeneScan users only Software must know where to put and or locate these files to operate the instrument and automate various tasks For example the selections displayed in the various pop up menus on sample and run sheets are located in these folders Typically these folders are located on the local hard disk The folder names and locations listed below are the ones specified during system installation The settings can be changed at any time If changes are made the new folder location and or name must be specified in the Preferences Folder Locations dialog box page 5 6 Location and Folder Folder Name Description Sample Sheet
253. iosystems Limited Warranty 0 6 Del Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com About This Instrument Chapter Contents In this Chapter The following topics are discussed in this chapter Topics See page Getting Started Quickly 1 2 What to Do If You Are a New User 1 3 Safety Information 1 4 Special Text Usage 1 5 User Bulletins 1 5 Technical Support 1 6 ABI PRISM 377 DNA Sequencer System Components 1 11 Theory of Operation 1 13 Using the Instrument 1 14 Data Analysis 1 18 Additional ABI PRISM Software 1 20 About This Instrument 1 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Getting Started Quickly To Get Started If you are not familiar with the ABI PRISM 377 DNA Sequencer follow the guidelines Quickly listed under What to Do If You Are a New User on page 1 3 If you are familiar with the instrument and its operation proceed as follows Before using the instrument read the safety information located on page 1 4 and in the ABI PRISM 377 Site Preparation and Safety Guide P N 903393 Ifyou are preparing your own gels proceed to Appendix A Gel Recipes for recommendations and instructions on preparing gel solutions Chapter 2 Pouring Gels for instructions on how to pour the gel Ifyou have a gel
254. itation Evaluate the quality and concentration of the DNA sample by Using the QuantiBlot Human DNA Quantitation Kit for human DNA Running an agarose yield gel If DNA is degraded or inaccurately quantitated reamplify with an increased amount of DNA Incorrect pH Verify buffer pH and concentration If correct quantitate sample DNA Too little or too much DNA can alter the pH Primer choice not optimal for example primers may be annealing to sites of template secondary structure or may have internal secondary structure Use different primers Refer to the GeneScan Reference Guide for more information Tm Of primers is lower than expected Decrease the annealing temperature by 2 C increments Troubleshooting 4 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Amplification Troubleshooting Guide Observation Possible Causes Recommended Actions Poor yield for multiplex PCR Non optimal thermal cycling parameters Between the denaturation and annealing stages add a 2 minute down ramp time to thermal cycling profile For multiplex PCR a short down ramp time is not necessarily optimal Competition from mispriming and other competing side reactions Use AmpliTag Gold DNA Polymerase Refer to the Refer to the GeneScan Reference Guide for more information on designing custom primers multiplexing PCR
255. ity Instrumentation Guaranteed 888 88 SOURCE www artisantg com Performing a PreRun and Loading the Samples Prepare the Loading Solution Flush the Wells Start the PreRun If not already prepared prepare the loading solution now Instructions are listed under Preparing the Formamide Blue Dextran Loading Solution on page 3 3 WARNING Tris borate EDTA TBE buffer can be harmful if inhaled ingested or absorbed through the skin It is irritating to the eyes skin and mucous membranes Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Before starting the prerun it is important to remove all the bubbles from the wells in the gel by flushing the wells with buffer To flush the wells Step Action 1 Fill the syringe needle attached with 1X TBE buffer from the upper buffer chamber 2 For shark s tooth combs only Starting at one end of the comb slowly inject the buffer across the well area to force out the air bubbles 3 Repeat this procedure until all air bubbles are gone Note Bubbles present during electrophoresis result in an uneven electrical field and poor electrical contact Bubbles will also electrophorese into the gel and alter the loading surface 4 If desired install the lid onto the upper chamber now However sample loading may be easier with the lid off See Using the Instrument in Chapter 1 for
256. ix 3 Under Source select Instrument file and then select the instrument file name The instrument file is displayed as shown below Copy Matrix For virtual filter set E all three matrix boxes will contain Source dRhod_BigDye H numeric values as shown Instrument here The numeric values will Comment be the same in each box Destination No Destination File Instrument Comment For virtual filter set A numeric X Copy Primer Matrix X Copy Taq Term Matrix values will typically be 1 000 0 12 0 011 0 000 1 000 0 12 0 011 0 000 i i i 0 455 1 000 0 183 0 000 0 455 1 000 0 183 0 000 displayed in the Primer and 0 248 0 483 1 000 0 151 0 248 0 483 1 000 0 151 Taq Terminator matrix boxes 0 115 0 282 0 529 1 000 0 115 0 282 0 529 1 000 only The numeric values will M Copy T Term Matrix not be the same in each box 1 000 0 12 0 011 0 000 0 455 1 000 0 183 0 000 a 0 248 0 483 1 000 0 151 Cancel 0 115 0 282 0 529 1 000 Note The numbers shown here are not representative values and will differ with each chemistry If the numbers in the matrix appear misaligned change the System Font from Charcoal to Chicago From the Finder choose Options in the Appearance control panel 4 Make sure the numbers in all the boxes range from 0 to 1 The numbers on the diagonals from top left to bottom right should all be 1 5 Click Cancel and quit the DataUtili
257. ket If a gap exists remove the plate and use your fingertips to flatten the gasket Then return to step 7 If a gap still exists remove the gasket clean off the sealant and return to step 1 Once there are no gaps remove the glass plate and proceed to the next step Glass plate INCORRECT CORRECT e GR1295 10 Carefully remove all excess sealant with a Kimwipe 11 Allow the sealant to cure for a minimum of 2 hours to overnight before use System Maintenance 8 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Procedure for The following is an illustration of the transparent upper buffer chamber Transparent Upper Buffer Chamber Removing the Old Gasket ELECTRICAL SHOCK HAZARD The ABI Prism 377 contains a high voltage power supply Although the instrument has been designed with safety features in the door to disconnect the power supply when the door is open please follow procedures as prescribed As with any electrophoresis apparatus be careful during instrument operation and when handling electrodes and liquids To remove the old gasket and sealant Step Action 1 Disconnect the electrophoresis cable from the instrument and remove the buffer chamber 2 Place the buffer chamber on a piece of clean lab paper so the lens is not scratched and does not touch the work surface 3 Using the spatula remove the old gask
258. l 2 3 Open the Fxn Name pop up menu and select Calibration File Send 4 Click Execute The CCD pixel position value and instrument serial number are sent to the instrument 4 28 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Temporary Loss of Signal Description Solution Gel Extrusion Description Solution This problem manifests itself as a band of little or no signal across the entire width of the gel image It usually occurs between 150 and 250 bases Temporary loss of signal has been traced to contaminants on the gel plates These contaminants include surfactants fatty acids and long chain polymers Rinsing glass plates in a dishwasher with hot deionized water 90 C has been found in most cases to remove the contaminants that cause temporary loss of signal In a few cases where a dishwasher did not work well soaking the plates overnight in a 5 solution of Multiterge detergent VWR Scientific P N 34171 010 eliminated the temporary loss of signal When voltage is applied on the ABI PRISM 377 DNA Sequencer the polyacrylamide gel sometimes moves from between the glass gel plates toward the cathode upper electrode and into the upper buffer chamber Up to about five centimeters of gel in a folded sheet can be deposited in the chamber This gel extrusion usually begins at the start of a run or even during the prerun
259. l Automated DNA Sequencing Chemistry Guide ABI 373 and ABI PRISM 377 DNA Sequencers P N 4305080 Also refer to the chemistry kit protocols and to our website at www appliedbiosystems com techsupport Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Data Collection Software Files and Folders Organizational The data collection software program and the various ancillary files and folders Structure required to perform runs generate sample files for data analysis and store data are organized inside two folders on the Macintosh hard drive ABI PRISM 377 Folder located directly on the hard drive ABI Folder located inside the System folder also referred to as the Settings Folder The folder and file structure is shown below Folders and Files Inside the ABI PRISM 377 Folder ABI Prism 377 a ff HEE Runs Sample Sheets Modules Chiller Modules Firmware Image 4G Prism 377 Collectio Files Inside the ABI Settings Folder System Folder i a Matrix B dR Inst File DP4 Ac 21M13 Matrix files Instrument files DyeSet Primer files Comb files The names of the ABI Folder files shown here are examples only and do not represent the entire contents of the ABI Folder Refer to the following tables for a description of the folders and files in the ABI PRISM 377 and ABI folders Data Collection Software 9 3 Artisan Technolog
260. l Control from the Window menu in data collection software 3 Open the Fxn Name pop up menu and select the CCD Pixel Position function The current pixel position value is displayed If it is the same as the value on the white label do not complete the remaining steps If the value is different continue Select the text box and type the correct CCD pixel position value Click Execute If the CCD Pixel Position error occurs again call technical support see Chapter 1 Using Calibration Use the Calibration File Make function from the Manual Control window to create the File Make ABI 377 Calibrations file and store the CCD pixel position value in it You can add the instrument serial number to the file as described below The ABI 377 Calibrations file is placed in the Preferences folder inside the System folder IMPORTANT You must have the correct CCD pixel position value in instrument memory before using Calibration File Make Creating an ABI 377 Calibrations File To create an ABI 377 Calibrations file Step Action 1 Turn power on to the instrument Select Manual Control from the Window menu in data collection software Open the Fxn Name pop up menu and select Calibration File Make 2 3 4 Click Execute and select Save from the File menu Adding the Serial Number to the ABI 377 Calibrations File You can add the instrument serial number to the ABI 377 Calibrati
261. l Loader Suppliers C 5 User s Manuals C 7 ABI PRISM DNA Fragment Analysis Kits and Reagents GeneScan Size Standards C 8 Fluorescent dNTPs C 8 Fluorescent dNTP PCR Kits C 9 Fluorescent Phosphoramidites C 9 Fluorescent NHS Esters C 9 Matrix Standard Sets C 9 Fluorescent Genotyping Demonstration Kits A and B C 10 ABI Prism Linkage Mapping Set Version 2 C 11 Part Number Updates Part numbers are subject to change Consult the Applied Biosystems World Wide Web Site www appliedbiosystems com techsupport for updated information Parts and Accessories C 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Glass Plates Spacers and Clamps cm Description Part Number 48 Stepped Glass Plate and Spacer Kit 48 cm 4305810 Includes two sets of 0 4 mm 48 cm well to read stepped glass for 96 lane plates and 48 cm spacers upgrade only Front Stepped Glass Plate 48 cm 4305387 for 96 lane upgrade only Glass Plate and Spacer Kit 401878 Includes two sets of 48 cm well to read glass plates and 48 cm spacers Rear Glass Plate 48 cm 401835 Front Glass Plate 48 cm 401838 Spacers two 48 cm 0 2 mm thick 401837 36 Stepped Glass Plate and Spacer Kit 36 cm 4305693 Includes two sets of 0 4 mm 36 cm well to read stepped glass for 96 lane plates and 48 cm spacers that must be cut to size before use
262. l pouring methods are located in Chapter 2 Pouring Gels Glass plates and all other gel pouring equipment must be ready for use prior to adding the polymerizing reagents to the gel solution Combine the following NmL 40 polyacrylamide stock 19 1 Note N is the desired gel percentage 144gurea 1 0 g mixed bed ion exchange resin 25mlL distilled deionized water Stir well until all the urea crystals have dissolved Use a 0 2 um cellulose nitrate filter to filter the polyacrylamide solution for 5 minutes Note During this step you both filter and degas the solution Add 4 0 mL of filtered 10X TBE and sufficient distilled deionized water to bring the total volume to 40 mL IMPORTANT If the plates are not clean and mounted in the gel cassette or other device clean and mount them now before adding the polymerizing agents to the gel solution Instructions are listed in Chapter 2 Pouring Gels Adding the polymerizing reagents Step Action 1 With the filtered polyacrylamide solution at room temperature add 200 uL of 10 APS ammonium persulfate Swirl gently to mix Be careful to avoid air bubbles Add 25 uL of TEMED to the solution Swirl gently to mix Be careful to avoid air bubbles WARNING TEMED tetramethylethylenediamine is a flammable liquid and is extremely corrosive Vapors may travel considerable distance to sources of ignition and flash back TEMED is
263. l your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment 7 EQUIPMENT DEMOS HUNDREDS OF InstraV ea REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED D a gaa tia Contact us 888 88 SOURCE sales artisantg com www artisantg com
264. lanes are contaminated Cancel the plate check fill the buffer chambers install the front heat transfer plate and start the prerun Prerun the gel approximately three minutes and watch the Scan window Contaminants may migrate out of the read region and peaks will disappear All lanes can then be used See pages 3 15 3 16 3 18 and 3 35 Click Cancel terminate the plate check and install a new gel Instrument Operation 3 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Skipping Lanes Determining Which The Scan window displays data as channel numbers Follow this procedure to Lanes to Skip determine the lane number s that correspond to the channel number s containing fluorescent contaminants Skip these lanes when loading samples Step Action 1 While the Scan window is open and the plate check is running hold a spare clean comb up against the scan window The comb must be of the exact configuration as the one used to prepare the gel Resize the Scan window to the exact size of the numbered lanes only on the comb a Scan Window E x 82 Y 6566 O Freeze Updates Current Scan Number 52 26 27 28 29 30 31 32 33 34 35 36 ELLELE LLLLL Click here and drag to resize the window The lanes in which the peaks appear are the lanes to skip when loading samples In the example sho
265. laser appear at the top of the window and move toward the bottom as new data is collected You can scroll up or down the Gel window to see parts that are not visible Remember however that this window is a re creation of the data through time and does not indicate the physical position of the fragments To display the Gel window choose Gel Image from the Window menu Gel File Image Window o 60 80 100 120 Scan 0 v End of run Data Collection Software 9 55 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The Electrophoresis The Electrophoresis History window displays the set and actual values for the History Window electrophoresis power supply and gel temperature throughout the course of a run The scale for each panel is adjustable The information in the Electrophoresis History window is also stored in the Gel file To display the electrophoresis history window open the Window menu and choose Electrophoresis History Electrophoresis History Double click here to adjust the scale Scan numbers To adjust the scale Step Action 1 Double click one of the panels in the window or click a panel once and choose Set Scale from the Edit menu The Set Scale dialog box is displayed Set Scale Maximum value 5 0 Minimum value 0 x Type in the minimum and maximum values you wish to display
266. le Optimize the hard disk when fragmentation is severe Check the level of fragmentation once a month using Norton Speed Disk IMPORTANT Always backup important files before optimizing the hard disk Optimizing the Hard Disk on page 8 19 Norton Utilities on page 8 22 Backup all programs and important files regularly Examples include Analysis and data collection software Matrix instrument files Customized files such as custom modules and analysis parameters Size standard files Backing Up Important Files on page 8 20 Do not add other software programs to the Macintosh that runs the instrument Adding Non Applied Biosystems Software on page 8 28 Install only one System file per hard disk System File on page 8 29 Configure SAM to scan the desktop only See SAM on page 8 21 Disable Norton s automatic optimization features See Norton Utilities on page 8 22 System Maintenance 8 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Restarting the Computer Overview Restart the Macintosh once each day or before every run Restarting the computer clears the random access memory RAM This helps ensure enough memory is available to begin data collection Rebuilding the Desktop Overview Rebuilding the desktop cleans up the Desktop s
267. le Module files are created and modified by opening an existing module file that has been selected on a run sheet How the file is saved determines whether a new module is created or an existing module is temporarily or permanently modified Note Rather than permanently modifying existing modules we strongly recommend using the modules supplied with the software as templates to create new modules This ensures you will always have all the modules designed for use with your instrument To modify an existing module or create a new one Step Action 1 Open the File menu and choose New 2 Click Sequence Run or GeneScan Run to open a new run sheet 3 Open the Run Module pop up menu and choose the module file you wish to work with 4 Click the small document icon next to the Run module pop up menu to display the Module Settings dialog box Click this icon to view the settings of the module selected Run Module GS PR 36F 2400v lt Data Collection Software 9 45 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To modify an existing module or create a new one continued Step Action 5 Enter new values in the respective fields as desired SE TETEE iii a Electrophoresis Voltage Collection Time Electrophoresis Current Gel Temperature Electrophoresis Power 50 Laser Power CCD Offset 0 CCD Gain
268. le 506 02 ccs eee set nese ercde eb eh wets ee baled aa deka 4 25 Using Calibration File Make and Send 0 0 0 cece teens 4 27 Temporary Loss of Signal sesse 66sec ce ee ntesa bebe Se eee eh eee eee eS 4 29 Gel Extr siof sisi ia Sue s hak due a gee dng A e iE O enaytidcban gees Ghee ane A 4 29 Performing an Alcoholic KOH Wash 00 00 cece cee eee eee eee 4 30 Performing a3 M HCl Wash 2 2 ee eiea nona d aa EAR e enna 4 31 Removing Gasket Marks from Glass Plates 0 0 cece eee eee 4 31 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Removing Coolant System Clogs 0 0 0 eee eee eens 4 32 Optimizing Electrophoresis Conditions for GeneScan Applications 4 34 Setting Preferences 0c ccc cece eee e eee eee s S L Chapter Contents sice ig ae eiiiai iiia daar e ete nk ae ey esd Ag ado Sea recent as Clans 5 1 What Are Preferences 62 286 vtea es eee He SOHO SG Mae oh de een SRN ee wel ee 5 1 Where are Preferences Stored 0 0 00 e ene E E 5 2 Viewing and Modifying Preferences 0 0 ccc eect eee nes 5 3 If the Preferences File Becomes Corrupt 0 0 00 e eee cee eee 5 4 Folder Location Preferences 0 0 eee ccc cece ene e a E 5 5 Default File Name Preferences 2 2 6 cee ce be ee rece eben ete ee bee eens 5 8 Sequencing Sample Sheet Preferences 0 cece 5 10 Sequencing Run Sheet Pref
269. le GS Run 364 24 v Sample Sheet SampleSheetExa v J D Gel s Matrix File A Primer Matrix v Figure 9 1 Auto analyze selected and deselected on a GeneScan run sheet Starting the Run To start the run or close the run sheet or Closing the Run Sheet If Then aon you are ready to proceed a Open the File menu and choose Save A dialog box with instrument operation showing the default run sheet name and the location where the file will be saved is displayed Change the file name and storage location now if desired Folder where run sheets are stored i Run Folder 5 17 98 1 06 Macintosh HD wy Eject Desktop New Save this document as Cancel Run 5 1 98 1 15 PM Run sheet file name b Click Save c Click the appropriate button on the run sheet to start a plate check prerun or run For more information on the run sheet buttons and other commands used for instrument operation see Commands Used to Perform a Plate Check PreRun and Run on page 9 39 you wish to close the run a Open the File menu and choose Close sheet b Click Save A dialog box showing the default run sheet name and the location where the file will be saved is displayed shown in previous step Change the file name and storage location now if desired c Click Save Data Collection Software 9 37
270. lean and mount them now before adding the polymerizing agents to the gel solution Instructions are listed in Chapter 2 Pouring Gels Adding the polymerizing reagents Step Action 1 With the filtered polyacrylamide solution at room temperature add 200 uL of 10 APS ammonium persulfate Swirl gently to mix Be careful to avoid air bubbles Add 25 ul of TEMED to the solution Swirl gently to mix Be careful to avoid air bubbles WARNING TEMED Tetramethylethylenediamine is a flammable liquid and is extremely corrosive Vapors may travel considerable distance to sources of ignition and flash back TEMED is harmful by inhalation contact with the skin and if swallowed Use only in a chemical fume hood When handling wear lab coat safety glasses and chemical resistant gloves Immediately pour the gel Allow the gel to polymerize for at least 2 hours before use Gel Recipes A 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Supplier Information Item Supplier 29 1 acrylamide stock solutions Amresco Inc PAGE PLUS stock solutions 30175 Solon Industrial Parkway Solon OH 44139 4300 800 829 2805 216 349 2805 Long Ranger Singel FMC BioProducts 191 Thomaston St Rockland ME 04841 800 341 1574 Mutation Detection MDE Gel Solution 207 594 3495 P N 50621 Long Ranger Concentrate 5 solution P N 50
271. leanup after a run 3 45 cold boot instrument function 3 50 collection time suggested times 3 31 combs inserting 96 lane comb 3 6 inserting shark s tooth 3 6 inserting square tooth 3 6 D data viewing Gel window 3 41 Scan window 3 41 data analysis designating Settings folder for auto analysis 3 52 See also automatic data analysis using the stop and analyze feature 3 40 E electrical shock hazard installing gel cassette 3 9 performing a plate check 3 11 electrode cables connecting lower buffer chamber 3 9 connecting upper buffer chamber 3 15 disconnecting 3 45 electrophoresis chamber illustrated 3 8 electrophoresis current function 3 49 electrophoresis off function 3 49 electrophoresis on function 3 49 electrophoresis power function 3 49 Index 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com electrophoresis volts function 3 49 ergonomic hazard sample loading hazards and recommendations 3 37 Execute button manual control mode 3 48 external cooler off function 3 50 external cooler on function 3 50 F files deleting to ensure adequate disk space for new data files 3 40 firmware downloading new image 3 50 file location 3 51 firmware file folder location and description of 3 51 folder locations folder names and locations 3 51 to 3 52 how to set 3 52 to 3 53 formamide chemical warning 3 3 formamide blue dextran using in empty wells 3 38 front hea
272. les are described in the table below Conventions Name Definition Seq Module is for ABI PRISM DNA Sequencing Analysis Software applications GS Module is for ABI PRISM GeneScan Analysis Software applications Plate Check Plate check module PR Prerun module Run Run module 12 36 or 48 The well to read distance of the gel plates in centimeters A B C D E or F The virtual filter set used by the module 1200 1200 scans hour approximately 100 bph for sequencing 2400 2400 scans hour high speed approximately 200 bph for sequencing CHILLER Modules designed for use with an external cold water bath 9 42 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The following examples show how to interpret a module name Module Definition GS Run 36D 2400 CHILLER GS GeneScan Run Run 36 36 cm well to read glass plates D virtual filter set D 2400 2400 scans hour CHILLER chiller module Seq PR 48A 1200 Seq Sequencing PR prerun 48 48 cm well to read glass plates A virtual filter set A 1200 1200 scans hour Plate Check A Plate Check plate check A virtual filter set A To View the Settings To determine the instrument settings specified by a particular module open the ina Module module settings dialog box from a run sheet inst
273. ling the Front Heat Transfer Plate 3 18 Setting Up the Software for a Run 3 19 Preparing a Sequencing Sample Sheet 3 19 Preparing a GeneScan Sample Sheet 3 24 Preparing a Run Sheet 3 28 Performing a PreRun and Loading the Samples 3 35 Starting and Monitoring the Run 3 40 Viewing the Log File 3 44 Cleaning Up After the Run 3 45 Analyzing the Data 3 46 Archiving and Printing Data from Runs 3 46 The Read Region 3 47 Manually Controlling the ABI PRISM 377 Instrument 3 48 Setting Folder Location Preferences 3 51 Instrument Operation 3 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Summary of Procedures for Performing a Run To Perform a Run To perform a run electrophorese samples on the instrument you will O ON OOP O Prepare the buffer and loading solutions before you start or as needed Clean the outside of the gel plates and mount them in the gel cassette Install the lower buffer chamber and gel cassette Perform a plate check Install the upper buffer chamber and fill both chambers with buffer solution Install the front heat transfer plate Set up the software by completing a sample sheet and a run sheet Perform a prerun and load the samples Start the run Materials Required but not Supplied Available from Applied Biosystems 4 Available from Major Laboratory Suppliers 25 mM EDTA with 50 mg mL blue dextran pH 8 0 P N 402055 Matrix standard
274. lly for your instrument However additional instrument files may be required See When to Make a New Instrument File on page 7 3 What Does the The instrument file normally contains Instrument File Contain Three matrices A comment field Aninstrument name field These can be seen in the Copy Matrix window in the DataUtility program A copy of this instrument file is attached to every gel file and sample file when these files are first created For this reason each computer on which you use the Sequencing Analysis program must have an instrument file in the ABI folder which is located in the System Folder IMPORTANT Due to slight variations in the filters of the ABI 373 instruments and the CCD cameras of the ABI PRiSM 377 and ABI PRISM 310 instruments the instrument file created for your ABI PRISM genetic analysis instrument is sub optimal for other ABI PRISM genetic analysis instruments If you analyze sample files on a different computer from the one that was used to collect data be sure to copy the correct instrument file s to the analysis computer 7 2 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com When to Make a New Instrument File Guidelines During system installation an instrument file is created specifically for your instrument If a valid instrument file exists in the ABI folder inside the System Folder o
275. located enough memory when launched File searching For example data collection software may not be able to locate a file it needs to open or save Or a file might be corrupted Hard Disk Any hard disk problem will result in an error message Check these areas if the Log file lists Macintosh errors Run Norton Utilities Disk Doctor to check the hard disk and correct recurring problems Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com About Troubleshooting Firmware About the Firmware Symptoms of Corrupt Firmware The firmware required to run the instrument is stored in memory on the instrument A copy of the firmware is also included as part of the data collection software and is stored as a file in the ABI PRISM 377 folder The firmware is downloaded from the computer to the instrument during system installation This occurs automatically when the instrument is turned on and data collection software is launched for the first time If the instrument is turned off between runs a battery backup keeps the firmware stored in instrument memory New versions of data collection software received after system installation can also include new firmware When installed on the computer and launched for the first time the software will ask if the firmware on the instrument should be upgraded A power outage can sometimes clear the instrument memory or the firmware can becom
276. ls gel formulation A 2 gel solution protocols A 19 to A 21 ingredients and run conditions A 11 protocol A 11 to A 12 polymerization APS reagent purity and associated problems A 5 how affects gel quality A 7 initiator concentration A 8 TEMED A 6 poor resolution caused by acrylamide impurity A 5 porous gel factor that affects rate of polymerization A 7 positioning pins checking cassette installation 3 10 cleaning afterarun 3 45 pouring fixtures for gels part numbers C 3 pouring gels See gel pouring methods power switch 3 8 preferences default file name preferences setting 5 8 to 5 9 defined 5 1 to 5 2 dye indicator 5 17 to 5 19 folder locations preferences setting 5 5 to 5 7 for GeneScan sample sheets 3 24 forrun sheets 3 28 for sequencing sample sheets 3 19 general settings setting 5 15 to 5 16 GeneScan run sheet preferences setting 5 13 to 5 14 GeneScan sample sheet preferences setting 5 12 if the preferences file becomes corrupt 5 4 Project Info BioLIMS setting 5 20 Project Info preference 3 19 3 25 sequencing run sheet preferences setting 5 11 to 5 12 sequencing sample sheet preferences setting 5 10 setting folder locations 3 51 to 3 53 9 5 to 9 6 viewing and modifying where they are stored prerun commands to perform 9 41 performing prerun and loading samples 3 35 to 3 39 purpose of 1 16 to 1 17 Primer Express software description of software 1 20 5 3 5 2 9 39 to Artisan Technolog
277. lue as is or enter a different value See the Note in the preceeding step Verify that the correct radio button for the type of chemistry being used is selected Dye Primer Taq Terminator or T7 Terminator If not select the correct button Type information in the Instrument and Comment text boxes e g the instrument serial number or name and the date the matrix was created Click the New File button and enter a descriptive name for the file in the dialog box Since instrument files are specific to instruments and chemistries use these to name the file e g lt instrument name gt BigDye InstFile Note Only alpha numeric characters the period the dash and the comma are permissible characters for instrument file names Click Save The dialog box closes and the instrument file is saved in the ABI Folder in the System Folder 10 Click OK to start the matrix calculation The calculation takes about one minute When the matrix is complete the message Make matrix successfully completed is displayed If an error message appears and the matrix is not made see Correcting Errors in Matrix Creation on page 7 22 11 Choose OK to close the Make Matrix dialog box or wait about 20 seconds for the dialog box to disappear 12 If you wish to add a matrix for another chemistry to the instrument file repeat steps 2 11 with the following exceptions a For step 8 c
278. lution 2 To prevent calcium and other mineral deposits from reoccurring refill the system with a solution of deionized water and 5 0 antifreeze Refer to Refilling the Water Reservoir in Chapter 8 System Maintenance for more information Troubleshooting 4 33 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Optimizing Electrophoresis Conditions for GeneScan Applications Introduction Definition of Resolution Modifying Run Time 4 34 Troubleshooting Optimizing electrophoresis conditions run time run voltage and run temperature for GeneScan applications can greatly improve data quality run to run precision and or throughput When selecting values for these parameters consider the following factors Range of fragment lengths Required degree of resolution Type of genetic analysis you will be performing For example does the application require native or denaturing conditions The preset electrophoresis parameters in the application modules are set to ensure the following Detection of all fragments in the typical size range permitted by the application For example microsatellite loci are rarely over 400 base pairs in length Acceptable run times Acceptable resolution The resolution R of two peaks in an electropherogram is defined as follows gt 0 5 x W Wz where the P are the peak positions measured below t
279. lution Graduated cylinder large 10X Tris Borate EDTA TBE stock solution pH 8 3 at ambient temperature Procedure for preparing 10X TBE in Appendix A Water deionized IMPORTANT If the pH of the 10X TBE stock solution is not 8 3 0 2 discard and use fresh solution Do not attempt to adjust the pH WARNING Tris borate EDTA TBE buffer can be harmful if inhaled ingested or absorbed through the skin It is irritating to the eyes skin and mucous membranes Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Prepare this solution before you start or as needed based on the procedures in this chapter To prepare the 1X TBE buffer solution Step Action 1 Pour 120 mL of 10X TBE stock solution into the graduated cylinder 2 Dilute with deionized water to a total volume of 1200 mL and mix well Preparing the Formamide Blue Dextran Loading Solution Materials Required Storage Recommendations Preparing the Loading Solution Formamide deionized pH gt 7 0 Deionization procedure in Appendix A 25mM EDTA pH 8 0 with blue dextran 50 mg mL We recommend storing the materials required separately Formamide store at 20 C Freeze thaw formamide stored in Eppendorf tubes a maximum of 5 10 times before discarding EDTA with blue dextran store at room temperature WARNING CHEMIC
280. lysis Software analyzes the raw data collected from each sample Sequencing Analysis This software Software Tracks gel files Extracts sample information from gel files Performs multicomponent analysis Applies mobility corrections Normalizes base spacing Baselines data Determines analysis starting points Calls bases gt gt gt gt ad Includes the following two utilities GelDoc removes tracking data from a gel file repairs file resources and removes and restores the image from a gel file DataUtility makes or copies matrices for instrument files You can reanalyze and edit the sequence data Also data files are in formats that you can use with commercially available or user generated programs on the Macintosh or on other compatible computers Refer to the ABI PRism DNA Sequencing Analysis Software User s Manual for specific information about the Sequencing Analysis software About This Instrument 1 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Additional ABI PRISM Software Genotyper Fragment Analysis Software GenoPedigree GenBase Primer Express 1 20 About This Instrument Genotyper software analyzes the data generated by GeneScan Analysis Software It enables you to analyze and interpret nucleic acid fragment size and quantitation data by converting it to user defined results specific to your genotyping studies For exampl
281. m Cleaning Instrument Accessories How To Thoroughly clean instrument accessories after each run as described below Cleaning Glass Plates Using a Dishwasher Accessory Method of Cleaning Glass plates We strongly recommend cleaning glass plates in a laboratory dishwasher with a hot deionized water rinse cycle See Cleaning Glass Plates below for more information Heat transfer plates Wipe with damp lint free towels and allow to air dry ona non scratch surface Positioning pins in Wipe with damp lint free towels and allow to air dry Gel or TBE electrophoresis crystals collect on them white cakey substance and can cause chamber arcing CAUTION Arcing is a luminous low voltage high current electrical discharge Arcing can severely damage the instrument Spacers and comb Clean with deionized water and allow to air dry Refer to Chapter 2 Pouring Gels for more information Note Do not clean spacers and combs with organic solvents These tend to adhere and do not evaporate well Residual solvent can prevent the gel from polymerizing right up against the spacer Do not mark spacers or combs with liquid markers such as Sharpie markers Gel cassette Wipe with damp lint free towel and allow to air dry Must be completely dry before the next run or arcing can occur See the Caution statement above Clean plates spacers and combs are critical for successful gel preparation and a s
282. mands are listed opposite the corresponding command on the pull down menu see the File Menu illustration on page 9 10 File Edit Instrument Window Commands in the pull down menus can be active or inactive Active commands are shown in bold print and can be selected by the user Inactive commands are shown in grey and cannot be selected at that particular time You will see inactive commands become active at various times throughout the procedures you perform Window Status lt This command is active and can be selected by the user Log Sample Sheet This command is inactive and cannot be selected at this Run time by the user Scan Gel Image Electrophoresis History Manual Control Preferences R About ABI Prism 377 Collection The Apple menu is a standard Macintosh feature It displays and provides access to the desktop accessories installed on the system The first item in the Apple menu About ABI Prism 377 Collection opens the data collection software splash screen which shows general information about the software including the version Click anywhere on the splash screen to close it Data Collection Software 9 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com File Menu dite Edit instrums New Open 0 Close W Save 38S Save As Save a Copy In Import Export Page Setup Print One Print
283. matically at the end of the run IMPORTANT If the pop up menu lists lt none gt only software cannot find the folder that contains the dye set primer files To correct this you must set the Settings Folder Preference to the ABI Folder See Setting Folder Location Preferences on page 3 51 If the file is the same for all remaining samples click in the column heading to select the entire column open the Edit menu and select Fill Down Otherwise select the appropriate DyeSet Primer file for each sample individually Instrument Undo i Cut copy lt ___ Click in this field to select Paste gt the entire column Clear Set Any Prim gt saem A lt none gt Click these boxes to FillDown 30 OREN POP Hp MENUS Set Scale Show Clipboard PI PI a Select an Instrument The instrument file must be the same for all the samples File To select the instrument file Step Action 1 Open the Instrument File pop up menu for the first sample and select the appropriate file You must select a file if you want the data to be analyzed automatically at the end of the run IMPORTANT If the pop up menu lists lt none gt only software cannot find the folder that contains the instrument files To correct this you must set the Settings Folder Preference to the ABI Folder See Setting Folder Location Preferences on page 3 51 Click in the column heading to selec
284. matography PerSeptive DNA PNA and Peptide Synthesis systems CytoFluor FMAT Voyager and Mariner Mass Spectrometers tsupport appliedbiosystems com Applied Biosystems MDS Sciex api3 support sciex com Chemiluminescence Tropix tropix appliedbiosystems com In the United States and Canada technical support is available at the following times Product Hours Chemiluminescence 8 30 a m to 5 30 p m Eastern Time Framingham support 8 00 a m to 6 00 p m Eastern Time All Other Products 5 30 a m to 5 00 p m Pacific Time In North America To contact Applied Biosystems Technical Support use the telephone or fax numbers given below To open a service call for other support needs or in case of an emergency dial 1 800 831 6844 and press 1 Product or Product Area Telephone Fax Dial Dial ABI Prism 3700 DNA Analyzer 1 800 831 6844 then press 8 1 650 638 5981 DNA Synthesis 1 800 831 6844 then press 21 1 650 638 5981 Fluorescent DNA Sequencing 1 800 831 6844 then press 22 1 650 638 5981 Fluorescent Fragment Analysis includes GeneScan applications 1 800 831 6844 then press 23 1 650 638 5981 Integrated Thermal Cyclers ABI PRISM 877 and Catalyst 800 instruments 1 800 831 6844 then press 24 1 650 638 5981 Artisan Technology Group Quality Instrumentation
285. meter files Before optimizing the hard disk Backing up all important files Making sure the original or a copy of all the system software is available should it become necessary to reinstall the software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com SAM Product Overview Symantec AntiVirus for Macintosh SAM is comprehensive virus prevention Recommendations detection and elimination software Computer viruses are programs that replicate themselves in the computer system They can spread throughout the programs on your hard disk or spread over a network eventually causing severe damage SAM performs the following functions Checks applications for viruses when they are run Checks Microsoft Word documents and Excel worksheets for macro viruses Checks floppy disks for viruses when inserted into the hard drive Checks files downloaded from the Internet a BBS or email gt gt gt OH Monitors the Macintosh for activity that might indicate the presence of a virus All of the software supplied with the ABI PRISM 377 DNA Sequencer is free of computer viruses However any additional software installed on the computer could have a virus We recommend Keeping SAM activated on the Macintosh at all times Setting up SAM so that it scans the desktop only Otherwise it may scan the hard drive while data is being collected resulting in lost data
286. meters GS Parameters folder inside the GeneScan Analysis software folder Size Standard GeneScan only GeneScan Size Standard Folder GS Standards folder inside the GeneScan Analysis software folder Data Collection Software 9 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Procedure To set folder location preferences Step Action 1 Open the Windows menu select Preferences and then select Folder Locations 2 Click the field that corresponds to the folder location you wish to set Page Preferences Folder Locations v sample Sheet Folder Macintosh HD ABI Prism 377 5ample Sheets _ CA Module Folder Macintosh HD ABI Prism 377 Modules CA Folder Containing Run Folders Macintosh HD ABI Prism 377 Runs Firmware File Folder Macintosh HD ABI Prism 377 Firmware Image L Settings Folder Macintosh HD System Folder ABI Folder L GeneScan Anal ysis Parameters Macintosh HD GeneScan 65 Parameters Folder CA GeneScan Size Standard Folder Macintosh HD GeneScan 0 65 Standards Folder For example click this field to set the Module folder location preference ox Using the menus in the dialog box locate and select the appropriate folder Select Module Folder ABI Prism 377 Y Macintosh Chill
287. mical resistant gloves and safety glasses when handling Stir for 30 minutes at room temperature Check that the pH is greater than 7 0 using pH paper If the pH is not greater than 7 0 decant the formamide into a beaker containing another 5 g of ion exchange resin and repeat 30 minute stirring at room temperature When the pH is greater than 7 0 allow the beads to settle to the bottom of the beaker Remove the supernatant formamide taking care not to disturb the beads Dispense the deionized formamide into aliquots of 500 yL and store for up to 3 months at 15 to 25 C Use one aliquot per set of samples Discard any unused deionized formamide A 10 Gel Recipes Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 29 1 Polyacrylamide Gels Protocol and Run Conditions Ingredients and Run Conditions Protocol for 29 1 Polyacrylamide Gels For 36 cm well to read runs 4 5 29 1 Polyacrylamide Gel 6 M Urea Ingredient For 50 mL Run Conditions urea 18 0g For runs at 1200 scans hr 40 acrylamide stock 5 625 mL Use standard 36 cm 1200 scans hr run deionized water 25 0 mL modules Mixed bed ion exchange 0 5g Increase run time to 9 hr resin For runs at 2400 scans hr Filter and degas the above ingredients before adding TBE Use standard 36 cm 2400 scans hr run modules 10X TBE 5 0 mL Inc
288. mide are neurotoxins Avoid inhalation and skin contact Wear gloves at all times and work in a fume hood when handling acrylamide solutions and use appropriate precautions to avoid inhalation of crystalline acrylamide Ammonium Persulfate APS APS begins to decompose immediately when dissolved in water The result is loss of activity Therefore it is vital that this chemical be prepared fresh daily since it affects the rate of polymerization and thus the gel properties Store solid APS at room temperature in an airtight container with desiccant to keep it dry Gel Recipes A 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com A 6 Gel Recipes Reagent purity and associated problems continued Reagent Problems That Can Occur When Fresh Reagents are Not Used TEMED N N N N tetramethylethylenediamine TEMED is by nature very reactive and prone to oxidation The oxidized form is yellow and less active Using oxidized TEMED slows gel polymerization time thereby significantly altering the gel characteristics Because it is hygroscopic this initiator gradually accumulates water which increases the rate of oxidation WARNING CHEMICAL AND FIRE HAZARD TEMED is extremely flammable and can be very destructive to the skin eyes nose and respiratory system Keep it in a tightly closed container Avoid inhalation and contact with skin eyes and cl
289. mple File Name Run sheets for sequencing analysis applications contain the following information Parameter Description Plate Check Module File containing the instrument settings for the plate check PreRun Module File containing the instrument settings for the prerun Run Module File containing the instrument settings for the run Collect time The length of time in hours that data will be collected Sample Sheet The sample sheet prepared for the run When selected the information from the sample sheet is imported to the sample information fields on the run sheet Can be set as Preferences See Setting Run Sheet Preferences on page 9 31 9 28 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com GeneScan Run Sheets Parameter Description Well to Read Distance The well to read distance of the glass plates in cm Instrument File Also referred to as a matrix file an instrument file is a set of mathematical matrices used to compensate for the spectral overlap that occurs between the dyes used together as a set The instrument file created by the service engineer during system installation is given the instrument serial number as its name Depending on the chemistries used you may have to create additional instrument files An instrument file must be selected for data
290. mples with specific lane positions It also stores information about the sample sheet and module associated with the run Run files are small enough to store on floppy disk Gel files contain the raw data acquired by the data collection program They are typically very large 20 70 MB Because Gel files are so large we recommend deleting them from the hard disk after you have obtained satisfactory Sample files You do not usually need to keep gel files once lane tracking is adjusted and sample files are created Use magnetic tapes removable cartridge drives or optical drives to archive gel files Gel files are too large to fit on floppy disks IMPORTANT Do not discard a gel file until lane tracking is adjusted if necessary and verified Log files provide a good record of your runs Individually Log files are small enough to archive on floppy disks Use floppy disks a magnetic tape drive a removable cartridge drive or an optical drive to archive sample files A Sample file is 150 200 KB in size depending on the length of the run A 1 4 MB high density disk holds about six files Archive Sample files once the channel selections tracking are correct For sequencing applications only Seq files show the base letter sequence only or a header depending on the format you choose You can open them from word processing programs and print them You can also save the files in several formats that can be read by other software programs
291. n Guaranteed 888 88 SOURCE www artisantg com A 12 Gel Recipes To prepare the acrylamide urea solution Step Action 1 Instructions for preparing glass plates and two gel pouring methods are located in Chapter 2 Pouring Gels Glass plates and all other gel pouring equipment must be ready for use prior to adding the polymerizing reagents to the gel solution Prepare all stock solutions per the appropriate list of ingredients on page A 11 Combine urea 40 acrylamide stock 25 0 mL deionized water and mixed bed ion exchange resin in a 150 mL beaker Stir the solution until all the urea crystals have dissolved Filter the solution through a 0 2 ym cellulose nitrate filter Degas for 2 5 minutes Note Degas time for all gels should be constant to ensure a reproducible polymerization rate for all gels Transfer the solution to a 100 mL graduated cylinder Add filtered 10X TBE buffer IMPORTANT Always remove the mixed bed ion exchange resin by filtration step 5 above before adding the TBE buffer Resin will destroy the effectiveness of the buffer Adjust the volume to 50 0 mL with deionized water IMPORTANT If the plates are not clean and mounted in the gel cassette or other device clean and mount them now before adding the polymerizing agents to the gel solution Instructions are listed in Chapter 2 Pouring Gels Adding the polymerizing reagents
292. n Guaranteed 888 88 SOURCE www artisantg com Refilling the Water Reservoir Procedure Check the water level in the reservoir once a week Refill the reservoir when it is between one third and one half full To refill the water reservoir Step Action 1 Turn the pump off if it is running IMPORTANT Do not remove the water reservoir while a run is in progress 2 Open the water reservoir compartment on the right side of the instrument by pulling gently on the door 3 Unscrew the water reservoir and remove it by pulling downward Place a paper towel under the tubes connecting the reservoir to the pump to catch any drips 5 Fill the reservoir approximately 3 4 full 750 850 mL with a solution of deionized water and 5 0 antifreeze any brand 6 Replace the reservoir and insert the tubes that connect it to the pump before screwing the reservoir into place 7 Screw the reservoir into place and close the compartment door System Maintenance 8 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Replacing the Upper Buffer Chamber Gasket Two Styles of Upper Buffer Chambers Parts and Tools Required 8 6 System Maintenance Your instrument will have one of two styles of upper buffer chambers The earlier version illustrated on page 8 7 P N 604078 is white with a convex window and is referred to in this section as t
293. n Importing To import information into a sample sheet Information Step Action 1 Export the information to be imported to the sample sheet into a tab delimited text file Format the file based on the following guidelines The file should contain only the information you wish to import It should not have a header The information imported to the first field in the sample sheet is everything up to the first tab in the text file Each row in the text file should contain the information for one row in the sample sheet Rows should ordered as you wish them to appear on the sample sheet Open the Edit menu and choose Select All to select all fields in the sample sheet Open the Edit menu and choose Import In the dialog box choose the file you wish to import AJ OJ NJ Click OK Exporting Exporting information from a completed sample sheet creates a tab delimited text file Information The format of this file is the same as the format discussed in step 1 of the import procedure listed above To export information from the sample sheet to a text file Step Action 1 Activate the sample sheet window by clicking in it Open the Edit menu and choose Export In the dialog box enter a name for the text file 2 3 4 Click OK The icon used for exported files is shown below sample export Data Collection Software 9 27 Artisan Technology G
294. n Instrument File for Virtual Filter Set E from Matrix Standards 7 10 Checking Instrument Files 7 16 Making an Instrument File from a Sample File 7 18 Storing and Backing Up the Instrument File 7 19 Adding or Replacing a Matrix in an Existing Instrument File 7 20 Correcting Errors in Matrix Creation 7 22 Terminology Matrix vs The terms matrix file and instrument file are sometimes used interchangeably In this Instrument Files chapter the term Matrix file refers files that must be created to properly analyze data for GeneScan analysis software applications Refer to Chapter 6 Making Matrix Files for GeneScan for more information Instrument file refers to the files that must be created to properly analyze data for sequencing applications Making Instrument Files for Sequencing 7 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Instrument File Overview Purpose Although the dyes in a set fluoresce at different wavelengths some overlap in the spectra still occurs To correct for this overlap when analyzing data a mathematical matrix is created for each dye set and is stored in a file called the instrument file The instrument file must contain a matrix for each chemistry run on the instrument During data analysis the appropriate matrix is applied to remove any spectral overlap During system installation an instrument file is created specifica
295. n Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The Log File A Log file is created automatically for each run when the run is started Log files contain a comprehensive chronological record of all error and status messages generated by the data collection program during a run This information includes Start and stop times of the run Instrument and Macintosh errors Each Log file is stored in its respective Run folder inside the Runs folder These files can be very large and only the last 32K of information can be viewed using the data collection program To view the Log file using the data collection software program open the Window menu and choose Log Use Microsoft Word or Simple Text to view the entire Log file stored in a Run folder The following is an example of the kinds of information stored in a Log file gt 2 20 98 5 04 07 PM Run Started gt 2 20 98 5 04 07 PM Operator for Sequencing Run bap gt 2 20 98 5 04 07 PM Run Module Sent Seq Run 364 2400 gt 2 20 98 5 04 08 PM EP Voltage 3000 volts gt 2 20 98 5 04 08 PM EP Current 60 0 m gt 2 20 98 5 04 08 PM EP Power 200 watts gt 2 20 98 5 04 08 PM Gel Temp S1C gt 2 20 98 5 04 06 PM Laser Power 40 0 mW gt 2 20 98 5 04 08 PM RunTime 3 50 hours gt 2 20 98 5 04 08 PM CCD Offset 0 gt 2 20 98 5 04 09 PM CCD Gain Z gt 2 20 98 5 04 09 PM CCD X Pixel Posi
296. n a beaker to verify polymerization SOLLYD TE A Z N lt k imag ie aaa a y Lool Unscrew the syringe Do not remove the luer fitting If using a Then shark s tooth comb a insert the straight edge of the comb to form one large well b Verify no air bubbles are trapped where the gel and comb meet square tooth comb a insert the teeth of the comb b Verify no air bubbles are trapped where the gel and comb meet Reinstall the clear brace and lock it in place Remove the gel injection fixture 10 Close the bottom cassette clamps to secure the bottom of the plates 11 Flush the gel injection fixture immediately with water to clean it Clean the syringe in a similar manner if it is to be reused Note Allow the luer fitting to dry completely before using it again 12 Cover the bottom of the plates with plastic wrap or damp paper towel to prevent the bottom of the gel from drying out 13 Allow the gel to polymerize the amount of time recommended by the gel solution protocol Pouring Gels 2 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Method 2 Pouring the Gel Using Unmounted Plates Materials Required Binder clips Eight medium size Three large size Comb Kimwipes Plastic wrap Raised level
297. n empty syringe or compressed air can be used 2 16 Pouring Gels Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To attach the gel injection device continued Step Action 9 Attach the gel injection fixture as follows Hold the fixture underneath the plates at a 45 angle Rotate the fixture up and onto the plates as shown below Close the cassette clamps to lock the fixture in place Simultaneously lock down both clamps on the fixture Verify that the tension springs lock the fixture firmly against the notches on the outside of the gel cassette see exploded view below oap Verify that the edges of the plates are flush against the rubber gasket inside the fixture Luer fitting No gap here indicates fixture is properly locked in Lock both clamps down simultaneously 10 Place absorbent paper on the table at each end of the plates to catch any gel solution that spills during injection IMPORTANT Do not raise the cassette by placing objects such as blocks or pipet tip boxes under the plates This can cause the gel to be thinner in middle Pouring Gels 2 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Method 1 Part 3 Pouring the Gel Using Mounted Plates Preparing the Gel Solution Using
298. n the sample sheet is used to identify data transferred to BioLIMS If this field is left empty the data transferred to BioLIMS is identified by the gel file name only Project names are defined prior to setting up sample sheets by opening the Windows menu and selecting Project Info under Preferences For more information and instructions see Chapter 5 Setting Preferences Instrument Operation 3 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Open a New To open a new sequencing sample sheet and enter sample names Action Open the File menu and select New leave the run window open Click the icon named Sequence Sample Enter sample names in the Sample Name column by clicking in the Sample Name field and typing the sample name Enter names in the exact order the samples will be loaded onto the gel The numbers to the left of the Sample Name column represent the gel lane numbers Leave fields blank that correspond to empty lanes IMPORTANT Each sample must have a unique name Limit sample names to 27 characters including the default characters Do not use colons slashes or symbols in sample names Note More text can be entered than is visible Text automatically shifts as the information is entered Use the keyboard arrow keys to scroll through long entries To import sample names from tab delimited text files follow the instructions listed in Chapter 9
299. n volume depends on the application and chemistry Refer to your Volumes protocol or the following guides as appropriate ABI PRISM GeneScan Reference Guide P N 4303188 ABI PRISM DNA Sequencing Chemistry Guide P N 4305080 For GeneScan Applications Number of Wells Load Volume pL 24 and 36 1 5 50 1 0 1 5 66 0 5 1 0 96 0 5 1 0 For Sequencing Applications Number of Wells Load Volume HL 18 0 75 2 0 36 0 75 2 0 48 0 5 2 0 64 0 5 1 5 96 0 5 1 0 Instrument Operation 3 39 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Starting and Monitoring the Run See Chapter 3 Instrument Operation for a description of the run and its purpose To Start the Run To start the run Step Action 1 Click Cancel on the Run sheet and then click Terminate to cancel the PreRun 2 Wait five seconds and then click Run The data collected throughout the run is stored in a Gel file A dialog box displaying the default gel file name and storage location appears You can change the name and storage location of the gel file now if desired Do not use colons slashes or symbols in gel file names Click Save The Scan window is displayed Note If a dialog box appears indicating there is not enough room on the hard disk to save the files created by a run delete files particularly Gel files
300. n your Macintosh computer you need not create one A new instrument file should be created if any of the optics in the instrument change either due to service or age Some specific situations that require a new instrument file are The CCD camera in the instrument is replaced Arunshows consistent and proportional pull up peaks indicating poor or incorrect spectral separation Pull up peaks appear as smaller peaks of one color directly under larger peaks of another Fluorescence and spectral overlap are affected by the gel used for the run You may need to make a new instrument file if the type of acrylamide or other gel reagents is changed If you need to replace a lost or damaged instrument file and you do not have a backup copy refer to the Automated DNA Sequencing Chemistry Guide ABI 373 and ABI PRISM 377 DNA Sequencers P N 4305080 This manual contains information on viewing and copying matrices You may want to try restoring the instrument file by following the procedures in this manual before you recreate the entire instrument file The Data Utility Program What it is Used For The DataUtility program has two main functions for users to make instrument files and to copy matrices from one instrument file to another The program is located in the Utilities folder within the Sequencing Analysis folder When creating instrument files the correct data file for each matrix standard must be entered in the correct bo
301. nder the bottle Unscrew the water bottle and properly dispose of the fluid or OIN Fill the bottle with 800 mL of cleaning solution and reattach the bottle to the instrument Open the Collection software if it is not already open Turn the pump on a Open the Window menu and select Manual Control b Open the Fxn Name pop up menu and select Pump On c Enter 51 in the Value box and click Execute Allow the solution to circulate for 15 to 30 minutes Turn the pump off a Open the Fxn Name menu and select Pump Off b Click Execute To flush the cleaning solution out of the instrument Step Action 1 Open the panel on the right side of the instrument to access the water reservoir 2 Unscrew the water bottle and properly dispose of the cleaning solution 3 Fill the bottle with 800 mL of deionized water and reattach the bottle to the instrument Turn the pump on a Open the Window menu and select Manual Control b Open the Fxn Name pop up menu and select Pump On c Enter 51 in the Value box and click Execute Allow the water to circulate for 1 2 minutes Turn the pump off a Open the Fxn Name menu and select Pump Off b Click Execute Repeat steps 1 6 three more times to ensure the cleaning solution has been removed To fill the water bottle Step Action 1 Unscrew the water bottle and properly dispose of the so
302. ned eae panded pha eee hee peat a AEA 1 14 Data Analysis 2 snc octiva dens dete vane fied doe a a eo based eee eles 1 18 Additional ABI PRISM Software 0 0 cee cece eee e ences 1 20 Chapter Contents oeseri dott heel aaa Seed ee ed heal dasa ieeehesashd cutee 2 1 Related Information in Appendix A Gel Recipes 0 0 0 cece eee eee eee 2 1 Selecting a Gel Formulation 00 cece eee eee eee tee een nka nis 2 2 Importance of Using High Quality Gels 0 2 eee eee 2 3 About the Gel Pouring Methods in This Chapter 0 00 0 cece eee eee eee 2 4 Before Using New Glass Plates 0 0 eect eens 2 5 Cleaning Glass Plates Spacers and Combs 0 0 cece eee eee nes 2 6 Method 1 Part 1 Mounting Glass Plates into the Gel Cassette 000 2 10 Method 1 Part 2 Attaching the Gel Injection Device 0 0 0 0 00008 2 14 Method 1 Part 3 Pouring the Gel Using Mounted Plates 0 2 18 Method 2 Pouring the Gel Using Unmounted Plates 00 0008 2 20 3 Instrument Operation oc icin sta cedan Seven dientacebian ee J L Chapter Contents 0cceiie2 ac bese cede pa han Seine ne eens eae aw wee oes ae a G 3 1 Summary of Procedures for Performing a Run 00 0 cee eee eee 3 2 Materials Required but not Supplied 2 0 0 eee eee eee nee 3 2 Preparing the 1X Tris Borate EDTA TBE Buffer Solution
303. nescence Tropix 1 800 542 2369 U S only or 1 781 271 0045 1 781 275 8581 Applied Biosystems MDS Sciex 1 800 952 4716 1 650 638 6223 About This Instrument 1 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 1 8 About This Instrument Outside North America Region Telephone Dial Fax Dial Africa and the Middle East Africa English Speaking and West Asia Fairlands South Africa 27 11 478 0411 27 11 478 0349 South Africa Johannesburg 27 11 478 0411 27 11 478 0349 Middle Eastern Countries and North Africa Monza Italia 39 0 39 8389 481 39 0 39 8389 493 Eastern Asia China Oceania Australia Scoresby Victoria 61 3 9730 8600 61 3 9730 8799 China Beijing 86 10 64106608 86 10 64106617 Hong Kong 852 2756 6928 852 2756 6968 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 758 8268 60 3 754 9043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 22358 2838 886 2 2358 2839 Thailand Bangkok 66 2 719 6405 66 2 319 9788 Europe Austria Wien 43 0 1 867 35 750 43 0 1 867 35 75 11 Belgium 32 0 2 712 5555 32 0 2 712 5516 Czech Republic and Slovakia Praha 420 2 61 222 164 420 2 61 222 168 Denmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9
304. ng Data from Runs 00 0 eee ee eee ee 3 46 The Read Regio is sche i sane cde ee tab ed ade a deeauinde aoe eae ee aaa be beeen 3 47 Manually Controlling the ABI PRISM 377 Instrument 0 000 e eee eee ee 3 48 Setting Folder Location Preferences 0 0 cee cece cece eee ees 3 51 Troubleshooting 0 0 0 ccc cece cece cece cece Fel Chapter Contents coe dave iad wc oe pare eb See teva ws 4 1 For More Troubleshooting Information 00 00 c eee eee eee eee 4 2 Gel and Gel Image Troubleshooting Guide 1 2 2 0 cece eee eee eee 4 3 Instrument Troubleshooting Guide 0 2 0 0 eee eee 4 6 Data Collection Software Troubleshooting Guide 0 0 0 e eee eee eee ee 4 10 Signal Troubleshooting Guide 0 0 eect eee 4 11 Amplification Troubleshooting Guide 2 0 0 eee eee nee 4 16 Resolution Troubleshooting Guide 0 0 0 2 eect eee eens 4 17 Sample Mobility Troubleshooting Guide 0 0 0 cee eee eee eee 4 19 About Troubleshooting Software 0 0 0 cece eee eens 4 20 About Troubleshooting Firmware 0 00 c cece cence eee ee eee 4 21 Performing a Single Reset 00 2 eect eect eee ee ee 4 22 Performing a Total Reset ei 4 256c ccecs cae ka age ne ee kd a dee ae ere ea ee Se deleted 4 22 Pertomming a Cold Boots 2 seire aed nera ba Se ATR ALIA Roa ea ag eee 4 23 Locating the Firmware Image File 0 0 eee ce eee 4 24 CCD Pixel Position Val
305. ng new plates 2 5 cleaning after a run 3 45 cleaning before loading gel to 3 5 cleaning by hand 2 6 to 2 7 cleaning in a dishwasher 2 6 decontaminating 4 30 to 4 31 gasket mark 2 5 identifying front and back 2 5 part numbers C 2 read region description of 3 47 recommended dishwashers 2 8 1 2 3 4 H hard disk ensuring space for automatically created files 3 40 optimizing 8 19 hardware description of system components 1 11 HCI wash performing 4 31 heat transfer plate rear cleaning after a run 3 45 See also front heat transfer plate 1 6 to 1 10 e mail address internet address 1 9 telephone hours 1 6 telephone fax 1 6 to 1 9 help 1 6 I IMPORTANT user attention word defined 1 5 importing sample sheet information 9 27 INHERIT software 1 21 injection device fixture for gel preparation 2 14 instrument components hardware 1 11 models available software 1 11 instrument files adding replacing matrices 7 20 to 7 21 backing up 7 19 color guide for data display 7 4 to 7 5 1 11 to 1 12 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com DataUtility program description of program 7 3 for Virtual Filter Set A 7 6 to 7 9 for Virtual Filter Set E 7 10 to 7 15 from a sample file 7 18 to 7 19 overview 7 2 storing and backing up 7 19 summary of chemistries 7 4 terminology 7 1 verifying instrument files 7 16 to 7 17 when to make new file 7 3 instrument manuals
306. no fluorescence Buildup of charge on the surface of the glass plate See Gel Extrusion on page 4 29 for more information Plates not rinsed free of Alconox Clean the plates with an alcoholic KOH wash page 4 30 or a 3 M HCI wash page 4 31 Rinse plates thoroughly with distilled deionized water Lane wide region s of fluorescence Contaminated loading buffer or sample Remake solutions co loaded with sample Troubleshooting 4 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Observation Severely bowed gel image Possible Causes Clamping bottom of gel plates Recommended Actions Do not clamp the bottom of the gel Note Use clamps of equal tension along the side of the gel The clamps must be placed over the spacers Gel extruded between plates into upper buffer reservoir See Gel Extrusion on page 4 29 for more information Clean the plates with an alcoholic KOH wash page 4 30 or a3 M HCI wash page 4 31 Red or green smearing on gel Appearance of donut shaped band with black central hole Gel dried out before running Too much sample loaded Before storing a poured gel wrap the gel ends with damp Kimwipes and plastic wrap Use gel within 2 6 hours after pouring Load less sample or remake sample with a higher dilution Gel wide region of migrating fluorescence can be of any color
307. nt sends raw sample data back to the computer This data is stored in a Gel file Data is then automatically or manually extracted from the gel file and analyzed by sequencing or GeneScan analysis software Input Files Information from the following files is used by data collection software to operate the instrument and identify and store the data collected These files are located in the ABI PRISM 377 Folder File Type Location Description Sample Sheet Sample Sheets Contain information used for Oder mide te sample identification and tracking ABI PRISM 377 folder data analysis Module or Chiller Modules or Chiller Contain instrument settings for the various plate checks Module Modules folder pre runs and runs that can be performed The modules inside the to be used for a run are specified on a run sheet ABI PRISM 377 folder Run Sheet Individual run Contain folders inside the Runs folder inside the ABI PRISM 377 folder Module designations and other pertinent information used to direct instrument operation and data collection Sample information imported from the sample sheet Locations valid only if folder location preferences are not changed See Setting Folder Location Preferences on page 9 5 and Chapter 5 Setting Preferences for more information Data Collection Software 9 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Sample
308. nu to set the Analysis Range Click Analyze Choose Results from the Windows menu and check each electropherogram by a Clicking 4 in the of Panels menu b Clicking 1 under Dye Samples c Clicking 1 on the Sample Files side of the Results window If Then each peak is one color with the other colors flat under it the matrix is good the other colors are not flat under the peaks as shown below Blue p Green the matrix is poor If Then the matrix is good Save the matrix file to the ABI folder the matrix is poor Redo the matrix by using different start and stop points If this does not improve the matrix data run new matrix standards Making Matrix Files for GeneScan 6 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Making Instrument Files for Sequencing Chapter Contents In this Chapter The following topics are discussed in this chapter Topic See page Terminology 7 1 Instrument File Overview 7 2 When to Make a New Instrument File 7 3 The Data Utility Program 7 3 Summary of Chemistries 7 4 Color Guide for Data Display Windows 7 4 Making an Instrument File for Virtual Filter Set A from Matrix Standards 7 6 Making a
309. nually controlling the installing upper buffer instrument 3 48 to 3 50 chamber 3 15 materials required but not loading gel into cassette 3 6 to supplied 3 2 3 7 matrix standard samples loading samples 3 35 to 3 39 automatic analysis 3 32 3 33 manually controlling the entering on GeneScan sample instrument 3 48 to 3 50 sheets 3 24 materials required but not entering on sequencing sample supplied 3 2 sheets 3 20 performing plate check 3 11 to loading recommendations for 3 13 GeneScan 3 24 loading recommendations for sequencing 3 20 requirements 3 2 performing prerun and loading samples 3 35 to 3 39 preparing 1X TBE buffer solution 3 3 module folder location and description preparing GeneScan sample of 3 51 sheet 3 24 to 3 27 preparing loading solution 3 3 O preparing run sheet 3 28 to 3 34 overlay strips preparing sequencing sample using 3 5 sheet 3 19 to 3 23 read region description of 3 47 setting folder location preferences 3 51 to 3 53 setting up the software an overview 3 19 skipping lanes determining which to skip 3 14 starting and monitoring the run 3 40 to 3 43 summary of procedures 3 2 viewing the Log file 3 44 L laser safety bar 3 18 safety warning 3 13 laser power function description of 3 49 laser run function description of 3 49 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com P pipetting ergonomic warning and suggestions 3 37 pla
310. o help ensure a robust matrix is produced we strongly recommend you follow these guidelines Leave at least one empty lane between non matrix standard samples and matrix standard samples Leave one empty lane between each matrix standard sample IMPORTANT Matrices are dye set instrument and run condition dependent As such matrices must be remade when any of these conditions change For more information refer to Chapter 6 Making Matrix Files for GeneScan v Lane number on the gel SampleName Sample A loaded in lane 1 Lane 2 left empty First matrix standard sample loaded in lane 3 Lane 4 left empty Matrix standard sample green lt _ _ Second matrix standard sample loaded in lane 5 Selecting The Project Name field is for BioLIMS software version 2 0 and up users only Project Names Otherwise the field can be left blank If a project name is not identified the data transferred to BioLIMS is identified by the gel file name only Project names must be defined prior to setting up the sample sheet as Project Info Preferences Refer to Project Information Preferences in Chapter 5 Setting Preferences for instructions and more information To select project names Step Action 1 Open the Project Name pop up menu for each sample and select the project name Project Name Project 1 Project 2 Project 3 If the P
311. ocol Note For accurate quantitation of human DNA samples use the QuantiBlot Human DNA Quantitation Kit P N N808 0114 Insufficient enzyme in reactions Use the recommended amount of enzyme Incomplete activation of AmpliTaq Gold DNA Polymerase Repeat amplification making sure to Hold reactions initially at 95 C for 10 15 minutes Use the recommended buffer Note Both buffer pH and buffer composition affect enzyme activation Note At temperatures gt 95 C the enzyme is susceptible to irreversible denaturation If you suspect insufficient activation increase the incubation time not the incubation temperature Too little sample DNA added to reaction Note This is especially critical in human identification experiments because sample quality is often poor Quantitate DNA and use the amount recommended in the protocol Note For accurate quantitation of human DNA samples use the QuantiBlot Human DNA Quantitation Kit P N N808 0114 Incorrect or suboptimal thermal cycler parameters Check protocol for correct thermal cycler parameters If the correct parameters were used they may need to be optimized for your specific application For example allow a linear increase in extension time with increasing cycle number or increase time at the denaturation plateau Troubleshooting 4 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOU
312. odes 8 12 to 9 7 to 9 8 8 13 replacing upper buffer chamber gasket 8 6 to 8 11 See Also Macintosh computer maintenance manually controlling the instrument 3 48 to 3 50 materials required but not supplied 3 2 matrix adding replacing in an instrument file 7 20 to 7 21 See alsomatrix files verifying an instrument file 7 16 matrix files creating a matrix 6 6 to 6 8 evaluating the quality of 6 3 to 6 5 purpose of 6 2 terminology 6 1 verifying new matrices 6 8 to 6 9 when to remake 6 2 Index 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com matrix standard samples automatic analysis 3 32 3 33 entering on GeneScan sample sheets 3 24 entering on sequencing sample sheets 3 20 loading recommendations for GeneScan 3 24 loading recommendations for sequencing 3 20 requirements 3 2 MDE gel list of gel recipes and recommendations protocol A 18 menu commands 9 9 to 9 13 Apple menu 9 9 Edit menu 9 11 File menu 9 10 Instrument menu 9 12 Window menu 9 13 mobility sample inconsistent reagent purity and associated problems A 5 inhibited by buffer impurity A 6 troubleshooting guide 4 19 module folder location and description of 3 51 modules 9 42 to 9 48 chiller modules description of 9 44 installing 9 44 to 9 45 list of modules 9 47 modifying and creating modules 9 45 to 9 46 naming conventions 9 42 to 9 43 standard modules 9 42 unused modules 9 46 viewing module
313. oduction An instrument file can be made from matrix standards as explained above or it can be made from a sample file This procedure requires fewer steps than running matrix standards however the matrix made from a sample file may not be as good as one made from matrix standards The quality of an instrument file made from a sample file depends on the quality of the sample file used The best samples to choose for making a matrix have approximately 25 each of A C G and T A good example of this is the pGEM DNA with the 21M13 primer that is included as a control in every Ready Reaction Sequencing Kit To create an instrument file from a sample file Action Before making the matrix verify that lane tracking is accurate Adjust if necessary Duplicate the unanalyzed sample file four times Use the Duplicate command from the File menu in the Finder The four copies will have the following names Sample name Sample name Copy 1 Sample name Copy 2 Sample name Copy 3 A gt ad Sample name Copy 4 These four sample file copies are used in the same way as the four matrix standard samples Making the Instrument File from a Sample File Step 1 2 3 For Filter Set A instrument files Follow the procedure Making an Instrument File for Virtual Filter Set A from Matrix Standards on page 7 6 Whenever the procedure indicates a specific matrix standard to be used follow the table below
314. of run performed most often Most preferences have factory set default values All preferences including the factory set defaults can be changed at any time Setting Preferences 5 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com List of Preferences The following is a brief description of each preference See Preference Description page Folder Locations Location of the folders on the hard disk in which the 5 5 following are stored sample sheets standard and chiller modules Run folders and files the firmware image general settings matrix instrument files GeneScan software analysis parameters and GeneScan size standard files Default File Names Naming convention used by data collection software to 5 8 automatically name the new files and folders created for each run Sequencing Sample User designated parameters on sequencing sample 5 10 Sheet Defaults sheets GeneScan Sample User designated parameters on GeneScan sample sheets 5 12 Sheet Defaults Sequencing Run User designated parameters on sequencing run sheets 5 11 Sheet Defaults GeneScan Run User designated parameters on GeneScan run sheets 5 13 Sheet Defaults General Settings Instrument name global serial number plate check 5 15 module communication port and minimum number of scans Dye Indicators Colors used to display data on the computer screen and to 5 17 print data
315. og box is displayed in which you can view and modify the current settings for that particular preference The preferences file is modified whenever you click OK in a preferences dialog box Detailed instructions for modifying each preference are located in this chapter Setting Preferences 5 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com If the Preferences File Becomes Corrupt Indicators Occasionally the preferences file becomes corrupted When this occurs you will either be unable to launch the data collection software or you will experience problems with the application Note If you launch the data collection software and a dialog box asking you to set folder locations preferences is displayed you need only to reset the folder locations preferences to continue See Folder Location Preferences on page 5 5 To Create a New To create a new preferences file Preferences File Step Action 1 If open quit the data collection software program Note Data collection software must not running while you perform this procedure Open the System Folder Open the Preferences folder Click on and drag the existing preferences file to the trash and empty the trash Depending on the type of ABI PRISM 377 instrument you have the preferences file will be named ABI 377 ABI 377XL ABI 377 96 or ABI 377 18 Launch the data collect
316. oints down access to the internal gel temperature control unit is closed off arrow pointing down TTT External valve with AAOOOOUUOODOOOOADODOOOOAIOOOOOOOOODOOONNOOOOOIOOOOOOOAOOMOMMININ 0 0 cN o MMM DOO OAOA OO VADANA DATON a tatata __ c MLIIUIUUIUULUULUIIT TT AOOONOOOOIOOOONOOONIOOONIOOONIOOO NA EOATE NONO O NONONO ONNIN GR0686 Figure 1 Rear view of the instrument with external water bath attached Subambient Temperature Operation B 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Water Bypasses the With the external water bath attached water passes directly into the front and rear Gel Temperature heat transfer plates in the instrument bypassing the gel temperature control unit Control Unit GRO969 Pi Figure 2 Instrument interior view with arrows showing water flow Dotted line shows instrument components used with the external water bath External Water Bath If the external water bath used has a heating capacity to 60 C the instrument can be Attached for run under standard operating conditions ambient temperature plus 10 C 60 C with Standard Operating the external water bath attached
317. ol is a flammable chemical and is irritating to the skin eyes respiratory system It can cause nerve and liver damage CNS depression nausea vomiting and headache Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Mix the solution well It will take at least 15 minutes for most of the pellets to dissolve Note This recipe is for a saturated solution so some pellets will remain Store the solution with the bottle capped tightly During storage the color of the solution will turn dark red brown The solution can still be used and is good for 1 year Place some uncolored absorbent towels or other covering in the hood to catch spills Place the gel plates on the towels with the inside surfaces facing up Note The plates should be nearly level so that the cleaning solution does not run off onto the bench Only the inside gel side surface of the plates need be cleaned though the outside surfaces can be cleaned similarly Pour approximately 15 mL of the cleaning solution onto the area of the plate where the gasket mark is Allow the solution to remain on the plates for 10 minutes CAUTION Longer times can harm the plates Repeat steps 2 and 3 Rinse thoroughly with deionized water Clean plates as usual Pouring Gels 2 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE
318. ology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Instrument Troubleshooting Guide Observation Possible Causes Recommended Actions A dialog box appears indicating there is not enough room on the hard disk to start a plate check prerun or run Hard disk is too full Delete files particularly gel files to create enough space If the files are important back them up first Delete files by dragging them to the trash and emptying the trash See Chapter 8 for more information The following message is displayed Could not complete your request because the CCD pixel position is incorrect CCD pixel position value erased from instrument memory Re enter the CCD pixel position value Follow the procedure Location of the CCD Pixel Position Value on page 4 26 The buffer level in upper buffer chamber is going down but no external leaks are visible Buffer is wicking over ears of the plates and leaking down spacers _ Ears Follow this procedure a Siphon the buffer out of the upper buffer chamber b Dry the corners of the notched area at the top of the front plate c Apply asmall amount of molten agarose to the corners of the notches d Refill the upper buffer chamber Buffer splashes and crystallizes around the read region Buffer poured into lower buffer chamber too quickly Fill the chamber to the top edge
319. olution during polymerization Gently stir gel solutions to avoid the introduction of air bubbles Handle the gel solution gently when casting the gel to avoid the introduction of air bubbles Keep the vacuum strength and time constant during the vacuum filtration step in the protocols provided in this Appendix for run to run reproducibility Perform vacuum filtering and gel casting with the solution at room temperature since cold solutions have a greater capacity for dissolving oxygen Use fresh high quality reagents Carefully follow the gel preparation protocols in this manual Gel Recipes A 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Air bubbles Age of the Gel A 8 Gel Recipes Factors that affect the rate of polymerization continued Factor Affect on Rate of Polymerization Initiator TEMED and APS initiate gel polymerization Concentration Too much TEMED or APS decreases the average polymer chain length and increases gel turbidity Too little TEMED or APS can slow polymerization to the point where oxygen enters the monomer solution and inhibits further polymerization The concentration of initiators in degraded TEMED and APS may be too low for the protocols presented in this manual Air bubbles can distort the sample path which affects lane tracking Pour gels carefully and gently so air bubbles never form If air bub
320. olution to a final volume of 50 mL OoN Oo Stopper the cylinder and mix the contents thoroughly Filter the solution through a 0 2 um cellulose nitrate filter oh h O Degas for 2 5 minutes and transfer the solution to a wide mouthed container Note Degas time for all gels should be constant to ensure a reproducible polymerization rate for all gels IMPORTANT If the plates are not clean and mounted in the gel cassette or other device clean and mount them now before adding the polymerizing agents to your solution Instructions are listed in Chapter 2 Pouring Gels Adding the polymerizing reagents Step Action 1 Add freshly made 10 APS and swirl carefully to mix without introducing air bubbles Note Be as accurate and reproducible as possible when making the 10 APS solution Significant variation in this reagent can produce changes in data quality Add TEMED and swirl carefully to mix without introducing air bubbles Immediately pour the gel Allow the gel to polymerize for 2 hours before using Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Long Ranger Gel Solution Protocols Overview To obtain the longest read length 5 0 Long Ranger from concentrate or Singel and 4 8 PAGE PLUS gels are recommended for 36 cm runs at 1200 scans hr only These gel formulations have not been shown to con
321. on Gel Recipes and Protocols for preparing the following gel solutions are located in Appendix A Protocols For Sequencing Runs Gel Recipes gt gt a a SSCP 19 1 polyacrylamide any percent 29 1 polyacrylamide 4 25 and 4 5 Long Ranger 4 75 and 5 0 PAGE PLUS 4 8 and 5 25 Mutation detection MDE gel for single strand conformation polymorphism A variety of gels can be used for sequencing runs on the ABI PRISM 377 DNA Sequencer Because conditions vary from lab to lab one gel formulation may work better than another We recommend testing each formulation and selecting the one that performs best 19 1 polyacrylamide gels are frequently used for sequencing applications The gel formulations that typically provide the longest read lengths are as follows Plate Size and Run Speed Gel Formulations Expected Read Length in Bases 1200 scans hr 4 75 Long Ranger concentrate or Singel gel forms 5 25 PAGE PLUS 36 cm well to read 4 5 29 1 polyacrylamide 650 800 WTR plates 5 0 Long Ranger concentrate 1200 scans hr or Singel gel forms 4 8 PAGE PLUS 36 cm WTR plates 4 5 29 1 polyacrylamide 550 700 2400 scans hr 48 cm WTR plates 4 25 29 1 polyacrylamide 750 900 For GeneScan Runs Gels used for GeneScan runs on this instrument are 2 2 Pouring Gels 5 0 Long Ranger 4 25 19 1 polyacrylamide Mutation detection MDE
322. on floppy disks Note If agel file is deleted the data can be reanalyzed but cannot be retracked Use floppy disks a magnetic tape drive a removable cartridge drive or an optical drive to archive sample files described on page 9 60 A 1 4 MB high density disk holds about six files A sample file is 150 200 KB in size depending on the length of the run Save a sample file when you feel confident that the channel selections tracking are correct for the sample The contents of any of the editable window can be printed for example sample sheets run sheets module settings dialog boxes Before printing certain parameters can be specified such as the paper size the orientation of the page and whether or not the print should be reduced from actual size To set up a page for printing choose Page Setup from the File menu and make the appropriate selections The appearance of the Page Setup dialog box depends on the printer you are using Refer to the manual for your particular printer for information on page set up To print the contents of a window Step Action 1 Make sure the printer is on and loaded with paper 2 Click the window you wish to print to make it active 3 Choose Print or Print One from the File menu Print One prints the window immediately bypassing the standard print dialog box If you choose Print you can set the number of copies and the pages that you want to print The appearance of the pr
323. on page 3 19 36 cm run Proceed to step 2 Open the six gel cassette clamps between the upper buffer chamber and the laser beam safety bar Make sure the laser beam safety bar is down and locked into position Carefully position the front heat transfer plate up against the glass plates so it rests on the laser beam safety stop CAUTION The front heat transfer plate is heavy and can be damaged if dropped or bumped against a hard surface Exposed metal on a damaged plate will cause arcing Arcing is a luminous low voltage high current electrical discharge that can severely damage the instrument Handle the plate with care Close the cassette clamps to lock the plate in position Attach the grounding strap to the heat plate ground connection Connect the water supply tubes to the quick disconnect fittings NI oO a Proceed to Setting Up the Software for a Run on page 3 19 Quick disconnect fittings for water supply tubes Grounding strap Front heat transfer plate 3 18 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Setting Up the Software for a Run Overview This chapter does not include a detailed description of the data collection software See Chapter 9 Data Collection Software for more information Before starting a run you must set up a sample sheet and a run
324. ons file using Simple Text which is provided with the Macintosh operating system To enter the instrument serial number Step Action 1 Open the System Folder the Preferences Folder inside it and the ABI 377 Calibrations file Select the series of question marks displayed and type the instrument serial number in their place The serial number is located on the back of the instrument ABI 377 Calibrations Se 7 7 7 Instrument Serial Number 00206 CCD X Skip Select Save from the File menu Close the ABI 377 Calibrations file Quit the SimpleText program Artisan Technology Group System Maintenance 8 15 Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Using Calibration Use Calibration File Send from the Manual Control window to send the CCD pixel File Send position value and instrument serial number to the instrument The serial number is included in all Sample files created by the instrument and can be viewed in the annotative view of the sample file This is useful for instrument identification particularly if you operate more than one instrument To send a Calibration file to the instrument Step Action 1 Turn power on to the instrument Select Manual Control from the Window menu Open the Fxn Name pop up Select Calibration File Send oa AJ OIN Click Execute 8 16 System Main
325. ontact Use an automated multi channel pipette loader Locate the instrument on a variable or predetermined height worktable or lab bench Use a stable stool or stepladder Install adequate artificial lighting in the appropriate area to facilitate loading Ensure adequate front access to instrument while performing loading activities Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Special Text Usage User Attention Words User Bulletins What are User Bulletins The text of this manual includes four user attention words designed to draw your attention to safety issues or issues relative to proper instrument operation Each represents a certain level of attention or action as described below Note Calls attention to information IMPORTANT Indicates information that is necessary for proper instrument operation or for effective chemistry CAUTION Indicates that damage to the instrument could result if you do not comply with this information WARNING Indicates that physical injury to the user or other persons could result if these precautions are not implemented User Bulletins are the mechanism we use to inform our customers of technical information products improvements and new products that are related to the operation of the ABI PRISM 377 DNA Sequencer and related laboratory techniques Examples of topics covered in user bulletins include New versions o
326. ontact with skin eyes and clothing Always work under a hood and wear chemical resistant gloves when handling TEMED solutions Urea Store at room temperature Ammonium APS is hygroscopic Store solid APS at room temperature in an airtight container Persulfate APS Store with desiccant if the environment is humid If tightly sealed it can be stored at 4 C for up to 1 year Gel Recipes A 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Index Numerics 10X TBE buffer solution 3 6 3 9 1X TBE buffer solution preparing 3 9 A acrylamide denaturing gels described 3 2 reagent purity and associated problems 3 5 safety warning 3 5 3 23 storing 3 23 air bubbles affects on gel quality 3 8 between comb and solution 3 8 Ammonium Persulfate See APS APS effect of changing concentration 3 8 storing 3 23 B BigDye primers kit 3 3 BigDye terminators kit 3 3 bis acrylamide safety warning 3 5 3 23 buffer solution how impurities affect reproducibility 3 6 preparing 1OX TBE 3 9 preparing 1X TBE 3 9 C chemistries chemical abbreviations used in gel recipes 3 9 sequencing gel recipes 3 3 contaminants of acrylamide 3 5 buffers 3 6 Tris 3 6 urea 3 6 D deionizing formamide 3 10 diffuse bands reagent purity and associated problems 3 5 dRhod
327. ook at the Autoanalyze with field Preferences Page Sequence Run Defaults Y Operator Mody Carrillo wTR 36 w cm Lanes 24 v PreRun Module Seq PR 36A 1200 Y Run Module Seq Run 364 1200 v Bel sutpene en With Analysis software E Auto Print Ee not selected Co If the analysis software is Then selected click OK to return to the run sheet not selected a Open the Autoanalyze with pop up menu and select Other b In the dialog box locate the ABI PRISM DNA Sequencing Analysis Software and click Open c Close the current run sheet and create a new run sheet Note Changing the preference has no affect on run sheets created prior to the change On the run sheet verify that the Auto Analyze boxes are selected for each non matrix standard sample If the boxes are not selected a Open the sample sheet by clicking the icon next to the sample sheet pop up menu on the run sheet Sample Sheet Sample Sheet 5 1_ 7 Click this icon Select a dye set primer file for each sample you want analyzed automatically Close and save the sample sheet Open the sample sheet pop up menu and select lt none gt Open the sample sheet pop up menu and reselect the sample sheet to update the information oap If matrix standard samples are being run deselect Auto Analyze for all matrix standard samples Select Auto Print
328. op See Rebuilding the Desktop on page 8 18 System Maintenance 8 25 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Downloading Data Collection Software from Our Website Overview The most current version of ABI PRISM Data Collection Software for this instrument can be downloaded from our website The procedure we recommend is as follows 1 on RwN Save the existing software under a new name This is recommended as a safeguard just in case something goes wrong when installing the new software Delete the existing Preferences file by saving it under a new name Download the new software from our website onto a floppy disk Load the new software from the floppy disk onto the hard drive Launch the new software Reset the Folder Locations preferences and any other preferences you use Renaming the During installation the new data collection software automatically overwrites the older Existing Software version currently installed on the hard drive We recommend saving the older version and Preferences under delete recom To ren a different name before the new version is installed The old version can be d once you have verified the new version is successfully installed We also mend deleting the existing Preferences file prior to installing the new software ame the old software and existing Preferences file Step Action 1 Rename the ABI PR
329. ore easily be tracked back to a particular set of plates Use an engraving pen to mark the glass near the top of the outside of each plate Make a small scratch with an engraving pen or place a small piece of waterproof tape near the top of the outside of each plate IMPORTANT Do not use a ballpoint pen or other liquid marker to identify plates Mark plates near the top as shown below to ensure that the identifier is not near the read region defined in Chapter 3 or seal area of the upper buffer chamber Locate the identifier at gt the top of each plate Always face the side Front plate with the identifier away Rear plate from the gel Ld Refer to Chapter 3 Instrument Operation for more information on the read region and upper buffer chamber Pouring Gels 2 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Cleaning Glass Plates Spacers and Combs Why Clean Plates are Important Using a Dishwasher Cleaning by Hand 2 6 Pouring Gels Clean plates spacers and combs are critical for successful gel preparation and a successful run Plates that are cleaned thoroughly and consistently will also help avoid the temporary loss of signal that can occur sporadically on this instrument Our research indicates this loss of signal is due to contaminant molecules surfactants fatty acids long chain polymers attached to the surf
330. othing Always work under a hood and wear chemical resistant gloves when handling TEMED solutions Urea Old urea has breakdown products that affect sample migration Guidelines for using urea are as follows Use urea in the crystalline form Weigh urea fresh each time it is used Prepare solutions containing urea fresh each time Do not prepare stock solutions containing urea WARNING CHEMICAL HAZARD Urea is a potential mutagen Avoid inhalation and contact with skin eyes and clothing Buffers and Other Gel Components Metals non buffer ions and decomposition products are contaminants commonly found in reagents such as Tris borate and urea Transition metal ions tend to affect gel polymerization in various ways causing run to run irreproducibility Non buffer ions tend to inhibit DNA mobility also causing run to run irreproducibility To help avoid these problems Use only high quality Tris borate and urea Use only distilled deionized water to dilute reagents Do not use 10X TBE buffer which has precipitated The change in ion concentrate affects sample migration Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Rate of The properties of the gel depend on the rate of polymerization The rate of Polymerization polymerization is affected by Temperature Oxygen Initiator concentration Each parameter is discussed in
331. ou to quickly organize and locate critical information However you choose to configure and implement this software system the integrity of your data is ensured One copy of the original sequence data is preserved at all times so the original data is never lost or written over not matter how often the data is analyzed and edited For further protection data backup and archiving utilities are built in Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The ABI PRISM BioLIMS system streamlines data retrieval and reporting by allowing you to locate specific sets of information Database queries can be performed using various data file attributes including project name sequence name instrument date and operator Your query results can be saved and printed for documentation and reference The power of this centralized database gives local and remote users immediate access to their data Whether you e in a different lab or working in your home you can use ABI PRISM BioLIMS query tools to quickly retrieve the information you need About This Instrument 1 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com ABI PRism 377 DNA Sequencer Pouring Gels Quick Start Guide BSS BibEystems Artisan Technology Group Quality
332. oup Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To prepare the work area and glass plates continued Step Action 6 Place the spacers on the rear plate as shown below notched side facing the middle of the plate Two to three droplets of water can be applied to the edge of the glass where the spacers will rest to keep them from moving IMPORTANT Align the outside edge of each spacer with the outside edge of the plate Spacers must cover the notched areas of the plate Place water droplets along edges of plate to hold spacers in position Spacers Bottom of rear plate Place the front plate on top of the rear plate with the gasket mark facing up hydrophobic area described on page 2 5 Align the bottom of the plates so they are flush Rear plate with spacers Front _ plate Attach four medium binder clips along the length of each side of the plates Evenly space the clips and clamp them so pressure is applied to the middle of the spacers IMPORTANT To avoid uneven gel thickness do not position binder clips any further onto the plates than the middle of the spacers ibs ribs lt Pressure from binder clip applied to middle of spacer GR1158 Slightly raise the end of the glass plates with the large notch in the front plate This will help the solution flow more easily to the bottom of the plates Pouring Gels 2 21 Artisan Technology
333. out This Instrument AutoAssembler can be upgraded to client server based INHERIT AutoAssembler for large scale sequencing projects With INHERIT any number of Macintosh clients are networked with a SUN Microsystems server and FDF Data Search System to provide shared use efficiency and faster performance The user interface for INHERIT AutoAssembler and AutoAssembler are identical Sequence Navigator is a two part software package designed for automated DNA and protein sequence comparisons It accurately aligns sequences to a standard or a population and identifies sequence variants To accomplish this Sequence Navigator integrates with data collected on the ABI PRISM 377 DNA Sequencer as well as text files from other sources Sequence Navigator comes standard with two powerful software modules Factura and Sequence Navigator Factura automatically identifies critical patterns as it sorts through and deactivates needless data Factura s automated batch processing for feature identification saves you time by recognizing features such as the vector primer and heterozygote IUB base positions Once sequence files have been processed in Factura and a batch work sheet created the work sheet is imported to Sequence Navigator With Sequence Navigator you can perform basic sequence editing use pair wise alignments to assemble contigs select multiple alignment to identify sequence variants and display the electropherograms collected by the
334. overview 1 18 to 1 19 9 60 See also automatic data analysis using the stop and analyze feature 3 40 Data Collection software description of 1 14 files and folders 9 3 to 9 4 menu commands 9 9 to 9 13 quitting 9 59 starting 9 8 troubleshooting guide 4 10 DataUtility program description of program 7 3 using to verify instrument files 7 16 to 7 17 deionizing formamide A 10 deleting data files 8 18 to 8 19 desktop how to rebuild 8 18 dew point table B 7 Documents on Demand 1 9 to 1 10 diffuse bands reagent purity and associated problems A 5 dishwasher recommendations 2 8 using to clean plates 2 6 dRhodamine terminator kit A 3 virtual filter used 7 4 dye indicator preferences 5 17 to 5 19 dye set primer files description of 9 49 how to select 9 50 naming conventions 9 49 to 9 50 dyes about fluorescent dyes 1 13 E Edit menu 9 11 electrical shock hazard installing gel cassette 3 9 performing a plate check 3 11 warnings in manual 1 4 4 32 8 2 8 7 8 10 8 12 electrode cables connecting lower buffer chamber 3 9 connecting upper buffer chamber 3 15 disconnecting 3 45 electrophoresis description of gel electrophoresis optimizing for GeneScan runs 4 34 to 4 35 electrophoresis chamber illustrated 3 8 electrophoresis current function 3 49 Electrophoresis History window 9 56 electrophoresis off function 3 49 electrophoresis on function 3 49 electrophoresis power function 3 49 electrophoresis volts function 3 49 elevate
335. oxes to indicate the dyes being run in each lane To deselect a color click in the appropriate box to remove the X Entering Sample The information in the Sample Info and Comments fields is imported into ABI PRISM Info and Comments Genotyper DNA Fragment Analysis Software and is used for sample identification and sorting If these fields are left blank only the words sample file will appear when sample information is displayed in Genotyper To enter Sample Info and Comments Step Action 1 To enter Sample Info click in the Sample Info field and type the information jm Sample Sheet for GeneScan LE GeneScan Sample Sheet Comments E Sample A blue comments es Sample A green comments E Sample A yellow comments EJ size standard comments qi F F 7 F Pla To enter a comment click the Comments field and type the information 9 24 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Saving and To save and close the sample sheet Closing the Sample Sheet Step Action 1 Do one of the following a Open the File menu and select Close b Click the box in the upper left hand corner of the window and then click Save Click Save A dialog box showing the default sample sheet
336. p Seq 7 0 9 0 36 2400 GS 2 0 4 0 2400 Seq 3 5 4 0 48 1200 Seq 10 0 11 0 29 1 amp LR 12 0 PP GS GeneScan application Seq Sequencing Application Note The number of scans that can be collected is limited by the software 2 If Then you do not want the data to be a deselect the boxes in the Auto analyzed automatically Analyze column by clicking in each box to remove the X b Proceed to Starting the Run or Closing the Run Sheet on page 9 37 this is a sequencing run and you want proceed to Auto Analysis for Sequencing the data to be analyzed automatically Runs on page 9 35 this is aGeneScan run and you want proceed to Auto Analysis for GeneScan the data to be analyzed automatically Runs on page 9 36 9 34 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Auto Analysis for See About Automatic Data Analysis on page 9 38 for more information Sequencing Runs IMPORTANT Do not analyze matrix standard samples To set up the run sheet for automatic data analysis Step Action 1 Verify that the data analysis software is selected in Preferences To do this a Open the Window menu and select Preferences b Select Sequence Run Defaults c Look at the Autoanalyze with field Preferences Page Sequence Run Defaults v Operator Mody Carrillo wTR _ 3
337. pacers Remove the gel using one of these methods Lay a large Kimwipe on the gel and roll it up Wash the gel off the plate with water d Properly dispose of the gel immediately do not contain a gel proceed to step 4 Wash the glass plates with a very small amount of detergent approximately 0 10 gram such as Alconox or Multiterge that is non fluorescent and will not leave a residue IMPORTANT Do not soak plates in detergent Use only a very small amount of detergent Detergent residue can cause red or other fluorescent fronts to appear on the gel image Thoroughly rinse the plates with hot water Rinse the plates again with hot deionized water IMPORTANT This step is critical for the complete removal of contaminants such as surfactants fatty acids and long chain polymers These contaminants can cause the temporary loss of signal during instrument operation Because the optimum water temperature is 195 F 90 C we recommend cleaning plates in a dishwasher to avoid injury Stand the plates up and allow them to air dry Lean plates so the side that will contact the gel is angled downward This will help keep dust and other airborne particles from settling on the inside surface of the plate Note If there is not enough time to let the plates air dry you can dry them gently with lint free towels such as Kimwipes Do not use compressed air to blow plates dry Never use an organic solvent to speed
338. parameters refer to Modifying and Information Creating Modules on page 9 45 in Chapter 9 Troubleshooting 4 35 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Setting Preferences Chapter Contents In This Chapter The following topics are discussed in this chapter Topic See page What Are Preferences 5 1 Where are Preferences Stored 5 2 Viewing and Modifying Preferences 5 3 If the Preferences File Becomes Corrupt 5 4 Folder Location Preferences 5 5 Default File Name Preferences 5 8 Sequencing Sample Sheet Preferences 5 10 Sequencing Run Sheet Preferences 5 11 GeneScan Sample Sheet Preferences 5 12 GeneScan Run Sheet Preferences 5 13 General Settings 5 15 Dye Indicator Preferences 5 17 Project Information Preferences 5 20 What Are Preferences About Preferences Preferences are default settings selected by the user in the data collection software Certain preferences such as the folder locations must be set to operate the instrument save and retrieve files and process the data generated by runs Other preferences are designed to save you time and customize the system For example you can reduce the time spent setting up sample and run sheets by changing certain sample and run sheet defaults to reflect the type
339. pen open the Windows pull down menu select Preferences and then select General Settings is open open the Page pop up menu and select General Settings 2 Set the preferences as follows a To enter an instrument name type the name in the Instrument Name field b To change the global serial number select the current value in the box and type in the new value use positive integers only c Do not select Suppress Left Right Averaging See explanation on previous page d To select a plate check module open the pop up menu and choose the appropriate module e To change the communication port click the appropriate port icon f To change the minimum number of scans select the current value in the box and type in a new value Preferences Page General Settings kal Instrument Name Jody Global Serial Number C Suppress Left Right Averaging Flate Check Module Communication Port Modem Printer No Port Minimum Number of Scans Note Data collection software collects data for the run time specified on the run sheet We recommend you do not enter a value below the default minimum number of scans 12000 If the value is less data collection software may not allocate enough hard disk space to collect all the data 3 If Then you are finished click OK to save your changes or Cancel to quit without making any changes you wish to change more preferences open the Page pop up menu and select another Preference 5 16 Setting Prefe
340. per 19 1 Acrylamide PP 29 1 Length incm Hour Application Gels Acrylamide Gels 12 1200 GS 1 0 1 0 12 2400 GS 1 0 1 0 36 1200 GS amp Seq 7 0 9 0 36 2400 GS 2 0 4 0 2400 Seq 3 5 4 0 48 1200 Seq 10 0 11 0 29 1 amp LR 12 0 PP GS GeneScan application Seq Sequencing Application Note The number of scans that can be collected is limited by the software 2 If Then you do not want the data to be a deselect the boxes in the Auto analyzed automatically Analyze column by clicking in each box to remove the X b Proceed to Starting the Run or Closing the Run Sheet on page 3 34 this is a sequencing run and you want proceed to Auto Analysis for Sequencing the data to be analyzed automatically Runs on page 3 32 this is aGeneScan run and you want proceed to Auto Analysis for GeneScan the data to be analyzed automatically Runs on page 3 33 Instrument Operation 3 31 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Auto Analysis for See About Automatic Data Analysis in Chapter 9 for more information Sequencing Runs IMPORTANT Do not automatically analyze matrix standard samples To set up the run sheet for automatic data analysis Step Action 1 Verify that the data analysis software is selected in Preferences To do this a Open the Window menu and select Preferences b Select Sequence Run Defaults c L
341. perature control system on and sets the temperature to a value between 20 and 60 C Pump Off Turns the temperature control system off CCD Pixel Position The charge coupled device CCD pixel position value is a reference point for alignment of the CCD camera with the laser beam The instrument is shipped with the correct CCD pixel position value in memory When a run is started data collection software checks for a value greater than zero If not found the error message shown below is displayed wy Could not complete your request because the CCD pixel position is incorrect If this occurs the value must be reentered See CCD Pixel Position Value in Chapter 4 Troubleshooting For convenience the value can be stored in a calibration file The value can be sent to the instrument using the Calibration File Send command See Using Calibration File Make and Send in Chapter 4 for more information Calibration File Make Used to create a file called ABI 377 Calibration that contains the CCD pixel position value and instrument serial number The file is stored in the Preferences folder inside the System folder See Using Calibration File Make and Send in Chapter 4 for more information Calibration File Send Used to send the CCD pixel position value and instrument serial number to the instrument The serial number is transferred into all Sample files created by the data analysis software
342. plates to dry Note Avoid other cleaning procedures or solutions that can reintroduce contaminants to the plates Removing Gasket Marks from Glass Plates Procedure An alcoholic KOH wash can also be used to remove buffer chamber gasket marks from the plates Step Action 1 Perform steps 1 5 above 2 Pour approximately 15 mL of the cleaning solution onto the area of the plate where the gasket mark is Allow the solution to remain on the plates for 10 minutes CAUTION Longer times can harm the plates Repeat steps 2 and 3 Rinse thoroughly with deionized water Clean plates as usual Troubleshooting 4 31 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Removing Coolant System Clogs Clog Indicators Recommendations Cleaning Solutions 4 32 Troubleshooting If tap water is used in the water reservoir calcium buildups can occur particularly in regions with hard water Calcium buildup can cause the following problems Flow switch sticks and plugs up High resistance to flow through heat plates Quick disconnect fittings leak due to buildup behind the seals Warning displayed that the pump has shut down during a run Poor resolution due to a gel running too cold accompanied by a warning that the pump has shut down Error message that the heat plate temperature exceeds 70 C even though the status window
343. quencing and one for GeneScan applications A sample sheet is a file that contains information used for sample identification sample tracking and data analysis A run sheet is a file that contains the sample identification tracking and data analysis information from the sample sheet the gel comb configuration and other pertinent information used to direct instrument operation during a run i e a plate check prerun and run module and to analyze data automatically after the run The data collection software is described in detail in Chapter 9 Data Collection Software 1 14 About This Instrument Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The Plate Check The purpose of the plate check is to ensure that the glass plates and gel are clean and free of fluorescent contaminants before the samples are loaded During a plate check the read region of the plates is scanned without electrophoresis The read region is the area of the glass scanned by the laser It is approximately 2 5 to 4 5 cm from the bottom of the glass It is 6 inches wide for standard and XL instruments 7 inches wide for 96 lane instruments and 3 inches wide for the ABI PRISM 377 18 instrument Top of glass plates 4 5 cm N 2 5 cm 4 Read region The number of channels in the read region are 194 for the standard ABI PRISM 377 and 377 18 instruments numbered 0 to 193
344. r Moving information from one lane to another To open a new sequencing sample sheet Step Action 1 If necessary launch the data collection software Instructions are listed under Launching the Data Collection Software Program on page 9 8 2 Open the File menu and select New 3 Click the icon named Sequence Sample 9 16 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Entering To import sample names from tab delimited text files follow the instructions listed Sample Names under Importing and Exporting Sample Sheet Information on page 9 27 Otherwise follow this procedure To enter sample names Step Action 1 Enter sample names in the Sample Name column by clicking in the Sample Name field and typing the sample name Enter names in the exact order the samples will be loaded onto the gel The numbers to the left of the Sample Name column represent the gel lane numbers Leave fields blank that correspond to empty lanes IMPORTANT Each sample must have a unique name Limit sample names to 27 characters including the default characters Do not use colons slashes or symbols in sample names Note More text can be entered than is visible Text automatically shifts as the information is entered Use the keyboard arrow keys to scroll through long entries 2 If matrix standard samples are being run ent
345. r Cancel to quit without making any changes you wish to change more preferences open the Page pop up menu and select another Preference Setting Preferences 5 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Sequencing Sample Sheet Preferences Setting Sequencing Two parameters can be set as preferences on Sequencing sample sheets Sample Sheet 4 DyeSet Primer Preferences Instrument File The factory default setting for both these parameters is none As such the DyeSet Primer and Instrument File must be manually selected each time a new sample sheet is created To save time preparing sample sheets set the defaults for these parameters to reflect the type of run most commonly performed To change sequencing sample sheet preferences Step Action 1 If the Preferences dialog box Then is not open open the Windows pull down menu select Preferences and then select Sequence Sample Sheet Defaults is open open the Page pop up menu and select Sequence Sample Sheet Defaults To change the default settings a Open the DyeSet Primer or Instrument File pop up menu b Select the desired file from the pop up menu Preferences Page Sequence Sample Sheet Defaults v e lt none gt DP4 Acv2 M1 3Rev DP4 Ac 21M13 DP4 Ac KS DP4 Ac M1 3Rev DP4 Ac Sk DP4sAc SP6 DP4 Ac T3 DP4 Ac T7 DP6
346. r beam The instrument is shipped with the correct CCD pixel position value in memory When you start a run data collection software checks for a value greater than zero If not found the following error message is displayed my Could not complete your request because the CCD pixel position is incorrect Perform the following procedures to locate and reenter the correct CCD pixel position value before using the instrument Locating and The correct CCD pixel position value is printed on a white label affixed to the CCD Entering the CCD camera The label is visible from the front of the instrument through the opening below Pixel Position Value the rear heat transfer plate Rear heat transfer plate CCD camera with pixel position label visible here To locate the CCD pixel position value Step Action 1 Have a flashlight available 2 Open the front door of the instrument 3 Shine the flashlight through the opening below the rear heat transfer plate and locate the white label on the CCD camera 4 Record the value on the white label 5 If you cannot find the label call technical support telephone numbers in Chapter 1 8 14 System Maintenance Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To enter the correct CCD pixel position value Step Action 1 Turn power on to the instrument 2 Select Manua
347. r changes in run conditions If you are using Virtual Filter Set C for GeneScan applications watch for evidence of matrix problems and remake the matrix as soon as problems appear 6 2 Making Matrix Files for GeneScan Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Evaluating Matrix File Quality How to Recognize Matrix Problems Bleedthrough Peaks or Pull ups A poor or incorrect matrix results in too much or too little subtraction of dye spectral overlap during data analysis Each causes a recognizable electropherogram anomaly Bleedthrough peaks also called pull ups caused by too little subtraction Elevated interpeak baseline caused by too much subtraction Bleedthrough peaks are small peaks of one color lying directly under a large peak of another color even though there is no PCR product corresponding to the smaller peak As shown Figure 6 1 the large peak signal is pulling up peaks in other colors Zm KDM01 82297 Beach n Display 2 5B 0595 7 GM sc 05 57 OW sr 05 57 BE ER 05 57 Xi Figure 6 1 Characteristic appearance of bleedthrough peaks Bleedthrough can occur for two reasons The matrix was made with the wrong dyes or filter set For a list of recommended dye filter set combinations refer to the GeneScan Reference Guide ABI 373 and ABI PRism 3
348. r gel solutions gently Filter pour gels at 20 23 C Variation in spacers Use spacers and comb sets of equal thickness Temperature of room gel solution or glass varies 20 23 C is optimal Slow mobility Total polymer concentration too high Bisacrylamide concentration too high Buffer concentration too low Check reagents Prepare new solutions using fresh reagents Old gel Pour a fresh gel and use within 2 to 6 hours of casting IMPORTANT Do not refrigerate Mobility too quick Total polymer concentration too low Bisacrylamide concentration too low Buffer concentration too high Check reagents Prepare new solutions using fresh reagents Note Do not use TBE buffer if it has precipitate in it Troubleshooting 4 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com About Troubleshooting Software Log File Error Macintosh computer operating errors are recorded in Log files A Log file is created Messages for each run and is stored in the corresponding Run folder The log lists error 4 20 Troubleshooting messages displayed during data collection including computer and instrument firmware errors Software related errors are usually due to problems with Memory For example the computer might not have enough memory to handle a certain function or the data collection program might not have been al
349. re the Settings folder must be designated as the ABI folder GeneScan Analysis Parameters Macintosh HD GeneScan Analysis X X GS Parameters Folder Contains the parameters used to analyze GeneScan run data and is located in the GeneScan Analysis software folder GeneScan Size Standard Foldert Macintosh HD GeneScan Analysis XX GS Standards Folder Contains the size standard definitions used to analyze GeneScan run data and is located in the GeneScan Analysis software folder IMPORTANT Locations and folder names listed are those at the time of system installation We strongly recommend not changing folder names and locations Displayed in Folder Locations dialog box for GeneScan users only Setting Folder To set the Folder Location preferences Location Preferences Step Action 1 If the Preferences dialog box Then is not open open the Window menu select Preferences and then select Folder Locations is open open the Page pop up menu and select Folder Locations 2 Click the box that corresponds to the folder location you wish to set SS Preferences Page Folder Locations X L Sample Sheet Folder C Macintosh HD ABI Prism 377 Sample Sheets WA CA Module Folder C Macintosh HD ABI Prism 377 Modules C Folder Containing Run Folders Macintosh HD ABI Prism 377 Runs A 7 Firmware File Folder Macintosh
350. re even peak heights in both dye terminator chemistries More intense signal with the BigDye primers and terminators Gel Recipes A 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Factors that Affect Gel Quality and Read Lengths A 4 Gel Recipes Factors One of the most critical variables that determines the success or failure of both sequencing and GeneScan analysis software runs is the gel The use of consistently prepared high quality gels will help ensure the best experimental results and minimize time spent troubleshooting gel problems Poor quality gels often cause problems that are mistaken for instrument problems For sequencing runs the quality of the gel directly effects the number of bases that can be called For GeneScan runs the quality of the gel effects the mobility of DNA fragments from run to run reproducibility of sizing signal strength and resolution Factors that affect gel quality include Purity and freshness of reagents Rate of polymerization Presence of air bubbles Age of the gel Other important variables that can significantly impact read lengths include gt gt gt Template quality base composition quantity Primer design Amount of sequencing reaction loaded on the gel Quality of formamide used in loading buffer Type of chemistry used Artisan Technology Group Quality Instrumentation
351. reRun and Run Module pop up menus are the files stored in this folder Chiller Modules Macintosh HD Contains the Plate Check PreRun and Run Folder ABI Prism 377 modules When setting up a run sheet the Chiller Modules selections displayed in the Plate Check PreRun and Run Module pop up menus are the files stored in this folder Folder Containing Macintosh HD Contains the Run folders A new Run folder Run Folders ABI Prism 377 is created for each run At the end of Runs analysis each new Run folder will contain a gel file log file sample files and a run file Firmware File Folder Macintosh HD Contains the firmware image file ABI Prism 377 Firmware Image IMPORTANT Locations and folder names listed are those at the time of system installation We strongly recommend not changing folder names and locations Displayed in Folder Locations dialog box for GeneScan users only Instrument Operation 3 51 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Folder Location and Folder Name What the Folder Contains Settings Folder Macintosh HD System folder ABI Folder Typically set as the ABI Folder The choices in other Preferences dialog boxes and in Run windows use the information contained in this folder for their pop up menus IMPORTANT To automatically analyze data after a run using the sequencing analysis softwa
352. ready for use prior to adding the polymerizing reagents to the gel solution Weigh out 18 0 g of urea and transfer it carefully to a graduated cylinder CHEMICAL HAZARD Urea is a mutagen Do not breathe the dust Refer to the Material Safety Data Sheet for the proper protective equipment that should be worn when working with this reagent Using a pipette add the following 5 0 mL 50 Long Ranger gel solution concentrate 5 0 mL 10X TBE WARNING CHEMICAL HAZARD Long Ranger gel solution contains acrylamide Acrylamide is a potent neurotoxin and is absorbed through the skin Effects are cumulative Always wear appropriate safety attire a full length laboratory coat protective glasses chemical resistant gloves etc and work under a chemical fume hood when handling powders solutions and gels Unpolymerized acrylamide sublimes Use in a well ventilated area and clean up spills immediately Slowly add distilled deionized water to bring the liquid level to approximately 45 mL Note Gently tap the cylinder while adding the water to release any air bubbles trapped by the urea Stopper and then invert the cylinder to dissolve the urea Note Although the cylinder and its contents become very cold the urea dissolves rapidly Allow the solution to warm to room temperature Add distilled deionized water to a final volume of 50 mL Stopper the cylinder and then mix the contents thoroughly OONO Use
353. rease run time to 4 hr 10 APS 250 uL TEMED 30 0 uL For 48 cm well to read runs 4 25 29 1 Polyacrylamide Gel 6 M Urea Ingredient For 50 mL Run Conditions urea 18 0g Use standard 48 cm run modules 40 acrylamide stock 5 31 mL Increase run time to 11 hr deionized water 25 0 mL Mixed bed ion exchange 0 5g resin Filter and degas the above ingredients before adding TBE 10X TBE 5 0 mL 10 APS 250 uL TEMED 30 0 uL WARNING CHEMICAL HAZARD Urea causes eye skin and respiratory irritation Lab experiments have shown mutagenic effects Avoid contact Wear chemical resistant gloves safety goggles and other protective clothing WARNING CHEMICAL HAZARD Acrylamide and bisacrylamide are neurotoxins Avoid inhalation and skin contact Wear gloves at all times and work in a fume hood when handling acrylamide solutions Use appropriate precautions to avoid inhalation of crystalline acrylamide WARNING CHEMICAL AND FIRE HAZARD TEMED is extremely flammable and can be very destructive to the skin eyes nose and respiratory system Keep it ina tightly closed container Avoid inhalation and contact with skin eyes and clothing Always work under a hood and wear chemical resistant gloves when handling TEMED solutions Read the MSDS in the Safety Summary included with your instrument user s manual Gel Recipes A 11 Artisan Technology Group Quality Instrumentatio
354. reference tables 4 3 to 4 19 removing coolant system clogs 4 32 to 4 33 resolution troubleshooting guide 4 17 to 4 18 sample mobility 4 19 signal troubleshooting 4 11 to 4 15 single reset performing 4 22 temporary loss of signal 4 29 total reset performing 4 22 U unmounted plates gel pouring method 2 4 upper buffer chamber See also buffer chamber filling 3 16 installing 3 15 replacing gasket urea contaminants A 6 effect of impurity A 6 safety warning A 6 user attention words 1 5 User Bulletins description of 1 5 user s manuals part numbers C 7 updates 1 5 8 6 to 8 11 V Virtual Filter Set A making instrument file 7 6 to 7 9 virtual filter sets about 9 51 Virtual Filter Set C making matrix files for 6 2 virtual filter sets about 9 51 Virtual Filter Set E making instrument file 7 10 to 7 15 virtual filter sets about 9 51 virus protection program description of program 8 21 WwW warnings acrylamide A 5 A 23 bis acrylamide A 5 A 23 safety information 1 3 TEMED A 6 A 23 urea A 6 WARNING user attention word defined 1 5 warranty D 1 waste nitric acid explosion warning 4 32 water reservoir refilling 8 5 wells flushing before loading samples 3 35 using formamide in empty wells 3 38 Window menu about 9 13 www address Applied Biosystems 1 9 Documents on Demand 1 10 part number updates C 1 1 9 to X XL Upgrade description of instrument 1 11 to 1 12 Index 9 Artisan
355. rences Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Dye Indicator Preferences Setting Dye Two categories of color definitions are available for the dyes used during a run Indicator Dye Color colors used to display data on the computer screen Preferences Plot Color colors used to print data Dye and plot colors can be changed at any time This feature is useful if you are color blind and the default factory settings are not appropriate for your needs To change Dye Indicator preferences Step Action 1 If the Preferences dialog box Then is not open open the Windows pull down menu select Preferences and then select Dye Indicators is open open the Page pop up menu and select Dye Indicators 2 To change the code used for a particular color type a different character in the appropriate field in the Code column Preferences Colors used to Page Bye Indicators vj display data on the computer Dye Color Plot Color 4 Colors used to Cate Ce print data BD Yellow v MB Black Y j E Red Y E Red Y j 7 Reset to Factory Settings Cancel 3 To change a color open the appropriate pop up menu and select a new color or select Other Setting Preferences 5 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88
356. rinted Data Collection Software 9 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Preparing a GeneScan Sample Sheet IMPORTANT Do not mix GeneScan and sequence analysis samples on the same sample sheet or in the same run Save Time by If the same type of run is performed repeatedly on the same instrument you can Setting Sample reduce the time spent setting up sample sheets by setting GeneScan sample sheet Sheet Preferences default preferences Setting sample sheet preferences means setting the default value of certain fields to the values used most often The following field can be set as a preference Std size standard dye color Once you have set this preference the preferred value appears automatically on each new GeneScan sample sheet This preference can be changed as often as necessary either by setting a new preference value or by manually selecting a different value on the sample sheet Instructions for setting GeneScan sample sheet default preferences are located in Chapter 5 Setting Preferences Entering See How to Enter Information on Sample Sheets on page 9 26 Procedures include Information Applying the same parameter to all fields in a column Copying information from one field or row to another Moving information from one lane to another Opening a New To open anew GeneScan sample sheet Sample Sheet Step Action 1 If neces
357. rnal water bath temperature at 3 4 C below the desired gel temperature Open the Status window and monitor the temperature Place a lid on the bath to minimize evaporation Add water and antifreeze during operation as needed to maintain the manufacturer s recommended water level Monitor the temperature and humidity in the lab see Avoiding Condensation Inside the Instrument on page B 7 CAUTION Condensation can cause decreased performance and severe damage to the instrument Monitor the room temperature and humidity consult the following condensation tables and maintain the water bath temperature setting above the corresponding dew point B 6 Subambient Temperature Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Avoiding Condensation Inside the Instrument How To Avoid Severe Operating the ABI PRism 377 DNA Sequencer at subambient temperatures may Instrument Damage cause condensation to form inside the instrument This can cause decreased performance and severely damage the instrument To avoid condensation keep the water temperature above the dew point temperature at which water vapor condenses into liquid or lower the humidity in the room Refer to the following table to determine the dew point of water at various relative humidities and ambient temperatures in your lab CAUTION Condensation can cause decreased performance and sev
358. roject Name is the same for all remaining samples click in the column heading to select the entire column open the Edit menu and select Fill Down Otherwise select the appropriate Project Name for each sample individually Data Collection Software 9 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Specifying the Size To specify the size standard dye color Standard Dye Color Step Action 1 For all non matrix standard samples designate the color of the size standard by clicking in the box to the right of the appropriate letter in the Color column B blue G green Y yellow R red When selected a diamond is displayed in the Std column This must be specified for data to be analyzed automatically Sample Name Project Name Color Std Pres Size standard dye color is red diamonds for matrix standard samples by clicking on the diamond Specifying the Dyes The dye colors run together in a lane must be specified for each sample Run in Each Lane To specify the dye colors run together in each lane Step Action 1 Click in the appropriate boxes in the Pres present column to select the dye colors that will be run in each lane for each sample Codes for the colors are displayed in the Color column B blue G green Y yellow R red Dyes be specified for data to be analyzed automatically Click in these b
359. roubleshooting Degraded primers Store primers in the dark at 15 to 25 C when not in use Avoid unnecessary freeze thaw cycles Keep primers on ice during run setup Expired or mishandled reagents Check expiration dates on all reagents If not expired verify that reagents are being stored and used according to manufacturer s instructions Compare with signal strength of the same samples prepared and run using fresh reagents Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Observation Possible Causes Recommended Actions Faint or no signal from sample DNA and from positive control continued Insufficient enzyme in reactions Use the recommended amount of enzyme Incomplete activation of AmpliTaq Gold DNA Polymerase Repeat amplification making sure to Hold reactions initially at 95 C for 10 15 minutes Use the recommended buffer Note Both buffer pH and buffer composition affect enzyme activation Note At temperatures gt 95 C the enzyme is susceptible to irreversible denaturation If you suspect insufficient activation increase the incubation time not the incubation temperature Too little sample DNA added to reaction Note This is especially critical in human identification experiments because sample quality is often poor Quantitate DNA and use the amount recommended in the prot
360. roughly clean surfaces subject to contamination Two methods can be used to clean spacers and combs Method 1 Clean the spacers and combs with hot water Then rinse them with hot deionized water and allow them to air dry Method 2 If the spacers and combs are very dirty clean them with a very small amount of Alconox Then rinse them with hot water followed by a hot deionized water rinse Allow them to air dry IMPORTANT Do not soak combs in detergent or even water for a long period of time such as overnight Soaking will break down the glue and the comb will eventually come apart 2 8 Pouring Gels Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Removing the An alcoholic KOH wash can be used to remove buffer chamber gasket marks from the Gasket Mark plates Preparation of all solutions should be carried out in a hood using safety glasses gloves and other appropriate protective clothing Step Action 1 Add 30 35 g of potassium hydroxide KOH or sodium hydroxide NaOH pellets to a plastic bottle WARNING Potassium hydroxide is hygroscopic and caustic It can cause severe burns and blindness if it comes in contact with the skin or eyes Always work in a fume hood Obtain a copy of the from the manufacturer Wear appropriate protective eyewear clothing and gloves Add 200 mL of absolute ethanol to the bottle WARNING CHEMICAL HAZARD Ethan
361. roup Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com About Run Sheets What is a Run Sheet A run sheet is a file that contains The software modules and other pertinent information used to direct instrument operation during a run A typical run consists of the Plate check Prerun Run See Chapters 1 and 3 for more information on the plate check prerun and run Sample identification information Gel comb configuration Information required for automatic data analysis see About Automatic Data Analysis on page 9 38 for more information The run sheet is also used to control the instrument In this context it is referred to as the run window The instrument is controlled by clicking the buttons at the top of the run window Plate Check PreRun Run Pause Resume and Cancel There are two types of run sheets one for sequencing and one for GeneScan analysis applications Sequencing The following is a new sequencing run sheet for standard ABI PRISM 377 instruments Run Sheets The run sheet for instruments with the XL or 96 lane upgrade differs slightly as noted in the following table Run 2 6 98 10 43 AM P erer gt re Plate Check Module PreRun Module Run Module lt none gt D0 Collect time hours Sample Sheet lt none gt v Well to Read distance _ 12 v cm Instrument File Operator Lanes v Lane Sample Sample Name Sa
362. roup Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Procedure To delete a file from the hard disk Step Action 1 If you wish to save the file store it on a floppy disk or other storage medium Drag the file into the trash can 2 3 Open the Special menu and choose Empty Trash 4 Click OK Optimizing the Hard Disk Why Optimize the Hard Disk Recommendations Before Optimizing the Hard Disk Over time files stored on the hard disk become fragmented stored in pieces in various locations on the disk As the amount of fragmentation increases computer performance slows because it takes the computer longer to find all the pieces Optimizing the hard disk Reduces fragmentation Frees up disk space Helps maintain optimum computer performance We recommend Checking the fragmentation level of the hard disk once a month using Norton Speed Disk Optimizing the hard disk when the fragmentation level is severe Turning off automatic optimization features Enabling these features can result in loss of data if the utility decides to optimize the hard disk while a run is in progress and data is being collected Refer to the Norton Utilities user s manual for instructions on using Norton Speed Disk Before optimizing the hard disk back up all important files onto floppy disks or another storage medium Make sure you know where all the original so
363. rrelation between the colors in the gel file and the base that each color represents IMPORTANT The gel file in the data collection program shows unconverted raw data so the colors displayed represent different bases depending on the chemistry 7 22 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com System Maintenance Chapter Contents In this Chapter This chapter describes how to maintain the instrument and computer The maintenance procedures in this chapter can be performed by users of the ABI PRISM 377 DNA Sequencer All other maintenance procedures should be performed by a trained Applied Biosystems service technician Topic See Page Instrument Maintenance Recommendations 8 2 Cleaning Instrument Accessories 8 3 Refilling the Water Reservoir 8 5 Replacing the Upper Buffer Chamber Gasket 8 6 Replacing Electrophoresis Cables and Electrodes 8 12 CCD Pixel Position Value 8 14 Computer Maintenance Recommendations 8 17 Restarting the Computer 8 17 Rebuilding the Desktop 8 18 Deleting Data Files 8 18 Optimizing the Hard Disk 8 19 Backing Up Important Files 8 20 SAM 8 21 Norton Utilities 8 22 Archiving Data from Runs 8 23 Reinstalling ABI PRISM 377 Software 8 25 Downloading Data Collection Software from Our Website 8 26 Adding Non Applied Biosystems Software 8 28 Sys
364. rs Inside the United States Supplier Supplier Headquarters Product Part No Kloehn 10000 Banburry Cross Dr Loader 18597 Company Las Vegas NV 89134 USA 0 25 mm Voice 702 243 7727 Loader 18663 Fax 702 243 6036 0 3 mm World Wide Web http Awww kloehn com Needle 18597 Outside U S offices are listed on the 0 25 mm 8 following page Needle 18628 0 3 mm 8 World Precision Sarasota International Trade Center Loader Gel Mate 96 Instruments 175 Sarasota Center Blvd Needle 67124 Inc Sarasota FL 34240 9258 USA 0 25 mm 10 Voice 941 371 1003 Fax 941 377 5428 World Wide Web http www wpiinc com Outside U S offices are listed on the following page Note Hamilton Co 702 858 3000 also supplies loaders that may work with this upgrade Suppliers Outside the US Supplier Supplier Contact Geographic Areas Served Kloehn Europe Bahnhofstrasse 12 Europe Postfach 55 CH 7402 Bonaduz Switzerland Voice 41 81 630 2303 Fax 41 81 641 3488 E mail kloehneurope bluewin ch World Precision P O Box 1191 Australia Instruments Inc Glen Waverly Victoria 3150 ihdo i Australia Australia Malaysia Voice 61 0 3 9887 6262 Fax 61 0 3 9887 9585 New Guinea E mail wpiau c031 aone net au New Zealand Parts and Accessories C 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Supplier Supplier Contact Geograph
365. rsions of software Plate Check Module Used to designate a default module for plate checks Communication Port The Macintosh computer port Modem or Printer to which the instrument is attached or No Port The Macintosh computer has two standard connection ports Modem and Printer Typically the instrument is attached to the Modem port and a printer or network connection is attached to the Printer port If however the instrument must be connected to the Printer port you must specify this change in the General Settings preferences window The No Port option allows you to use the data collection software when no instrument is attached to the computer or the instrument is turned off Minimum Number of Scans Determines the amount of hard disk space software sets aside for the gel file at the beginning of every run The default factory setting is 12000 If necessary this number can be increased We strongly recommend you never decrease this number If decreased the Macintosh may not have enough hard disk space to store the additional data Setting Preferences 5 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To Change General To change general settings preferences Settings Preferences Step Action 1 If the Preferences dialog box Then is not o
366. rtisantg com Setting Folder Location Preferences Overview The folder locations for the following types of files and folders must be designated to perform a run and to handle the data generated during a run Sample sheets Module files Run folders Firmware image file Settings gt gt gt gt o o GeneScan analysis parameters for GeneScan users only GeneScan size standards for GeneScan users only Software must know where to put and locate these files to operate the instrument These folders are typically located on the hard disk of the Macintosh attached to the instrument The folder names and locations listed below are the ones specified during system installation The settings can be changed at any time However if changes are made the new folder location and or name must be specified in the Preferences Folder Locations dialog box page 3 52 Refer to Chapter 5 Setting Preferences and Chapter 9 Software for more information Location and Folder Folder Name What the Folder Contains Sample Sheet Folder Macintosh HD Contains all the sample sheets When ABI Prism 377 setting up a run sheet the selections Sample Sheets displayed in the pop up menu for sample sheets are the files stored in this folder Module Folder Macintosh HD Contains the Plate Check PreRun and Run ABI Prism 377 modules When setting up a run sheet the Modules selections displayed in the Plate Check P
367. ructions listed below The module settings window shows the following information Electrophoresis Voltage Collection Time Electrophoresis Current Gel Temperature also referred to as the run temperature Electrophoresis Power Laser Power CCD Offset CCD Gain To open a Module Settings dialog box Step Action 1 Open the File menu and choose New 2 Click Sequence Run or GeneScan Run to open a new run sheet 3 Open the Run Module pop up menu and choose the module file you wish to view 4 Click the small document icon next to the Run module pop up menu to open the Module Settings dialog box R n Hodulel 6S PR 3EF 2A00 7 D Click this icon to view the settings BEETA of the module selected Data Collection Software 9 43 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To open a Module Settings dialog box continued Step Action 5 The Module Settings dialog box also allows you to change module settings Module settings can be modified temporarily for one run without changing the original module You can also use existing modules as templates for creating new modules or you can change the default settings of an existing module For more information and instructions see Modifying and Creating Modules on page 9 45 Electrophoresis Yaltage Collection Time Hours Electrophoresis Current Gel Temperature C Elec
368. s Power bas Laser Standby Laser Run Laser Power Pump On Pump Off Calibration File Make Calibration File Send CCD Gain CCD Offset CCD Pixel Position External Cooler On Relay 4 External Cooler Off Relay 4 Cold Boot Instrument 3 If appropriate enter a number in the Value field Guidelines are shown in the Range box and in the following table Per the guidelines shown in the Range field enter a value between 0 and 60 Fxn Fxn Name Range s5 Pump on v 0 to 60 C 4 Click Execute If necessary click Cancel to terminate a function 3 48 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Manual Control The following tables describe the functions that can be performed from the Manual Functions Control window Function Name Description Electrophoresis On Starts electrophoresis Electrophoresis Off Stops electrophoresis Electrophoresis Volts Sets electrophoresis volts to a value between 0 00 and 5 00 kV Electrophoresis Sets electrophoresis current to a value between 0 00 and 60 0 mA Current Electrophoresis Sets electrophoresis power to a value between 0 and 300 W Power Laser Standby Sets the laser to minimum power Laser Run Turns the laser on Laser Power Sets laser power to a value between 0 00 and 40 0 mW in 10ths of mW Pump On Turns the tem
369. s analyzed is often referred to as raw data The file in which the raw data is stored is referred to as a gel file The raw data of the gel is a matrix of information from the number of channels across the gel 194 388 or 480 in four colors multiplied by the number of scans collected The gel file contains an image of the data for display and the raw scan data Gel files range in size from 20 MB to 40MB At the end of data collection the analysis software ABI PRISM DNA Sequencing Analysis or ABI PRISM GeneScan Analysis software is used to manually or automatically process analyze and translate the collected data into either base sequence fragment sizing information or relative concentrations About This Instrument 1 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Using the Instrument Overview The process of setting up the instrument loading samples onto the gel and electrophoresing the samples is commonly referred to as performing a run An overview of the steps involved in performing a typical run are listed in the table below Step by step instructions are listed in Chapter 3 Instrument Operation Step Action 1 Set up the instrument 2 Perform a plate check 3 Configure the data collection software 4 Perform a prerun and load the samples 5 Start the run The term run is also used when referring to a run module described below
370. s fields is imported into ABI PRISM and Comments Genotyper DNA Fragment Analysis Software and is used for sample identification and sorting If these fields are left blank only the words sample file will appear when sample information is displayed in Genotyper To enter Sample Info and Comments Step Action 1 To enter Sample Info click in the Sample Info field and type the information EEE Sample Sheet for GeneScan z GeneScan Sample Sheet Sample Name Project Name Color Std Pres Sample Info Sample A Project 1 Sample A blue comments Sample A green comments size standard comments Sample A yellow comments To enter comments click in the Comments field and type the information 3 26 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Lane numbers Samples loaded v in lanes 1 and 2 Empty lane between non matrix and matrix samples Matrix sample loaded inlane 4 Empty lane between matrix samples Matrix sample loaded in lane 6 Project names must be predefined in Preferences Can be set as a Preference Sample Sheet SampleSheetExanp ee ees 4 GeneScan Sampl heet Project Name Color Std Sample Info Comments Project 1 First Sample Name First Sample Name First Sample
371. s too far into the gel Old buffer Make 1X fresh daily Do not use 10X TBE which has precipitated Old gel Use gels within 2 6 hours of casting IMPORTANT Do not refrigerate Gel not allowed to polymerize long enough Allow gel to polymerize the full length of time recommended by the protocol Gel stored under in bright light Do not store gel under or in bright light Variation in spacers Use spacers and comb sets that are of equal thickness Temperature of room gel solution or glass too warm or cool during polymerization 20 23 C is optimal 4 18 Troubleshooting For GeneScan applications only see also Optimizing Electrophoresis Conditions for GeneScan Applications on page 4 34 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Sample Mobility Troubleshooting Guide Observation Possible Causes Recommended Actions Inconsistent mobilities from gel to gel Total polymer wrong Follow protocol carefully Wrong concentration of Bis Follow protocol carefully Wrong buffer concentration Follow protocol carefully Do not use 10X TBE which has precipitated Poor quality reagents or old urea Remake the 10X TBE and acrylamide stock solution using highest grade reagents Use ultra pure urea Dissolved O concentration Keep vacuum strength time constant Stir pou
372. s used to determine the optimum sequence order resulting in a precise consensus sequence Multiple projects are efficiently managed and easily updated Convenient sequence and project reports monitor sequence quality and project progress In addition data integrity is always maintained because AutoAssembler allows you to go back and review all stages of your project at any time No data is ever lost and original sequence information is always preserved Deactivated vector and ambiguity sequence regions as well as edited base calls can be viewed or reactivated as original primary base sequences Moreover unlike other software programs this information is preserved within the original sequencer data files making data management easier and more reliable Results can be graphically viewed and quickly interchanged as project layouts bar and sequence alignments and aligned multiple four color electropherograms allowing you to analyze your data from several different perspectives The statistics view helps you quickly identify ambiguous sequence regions Electropherograms features tables sequence annotations and edited and primary sequence data are accessible and easily edited directly from within any part of the program and for any sequence About This Instrument 1 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Sequence Navigator ABI PRISM BioLIMS Software System 1 22 Ab
373. samples may not be required refer to your protocol and Chapter 6 Making Matrix Files for GeneScan or Chapter 7 Making Instrument Files for Sequencing as appropriate for more information Sequencing standard for sequencing runs only may not be required refer to your protocol for the recommended sequencing standard Size standard required for GeneScan analysis software runs only refer to your protocol for the recommended size standard Thermal cycler we recommend the GeneAmp PCR System 9700 or 9600 Thermal cycler accessories MicroAmp tray retainer sets reaction tubes and caps Formamide deionized see Appendix A for deionization procedure Gel freshly prepared recipes in Appendix A Gloves disposable powder free Kimwipes Microcentrifuge or centrifuge adapted for spinning microtiter plates Pipet and tips small volume calibrated Gilson Pipetman Rainin Instruments P N P10 or P20 Flat pipet tips Rainin Instruments P N GT 1514 Note Pipet tips listed above fit both the P10 and P20 However they eject off the P10 only Syringe 60 cc with a 16 to 18 G needle Tris Borate EDTA TBE stock solution 10X pH 8 3 Water sterile deionized 3 2 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Preparing the 1X Tris Borate EDTA TBE Buffer Solution Materials Required Preparing the 1X TBE Buffer So
374. san Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Auto Analysis for See About Automatic Data Analysis on page 9 38 for more information GeneScan Runs IMPORTANT Do not analyze matrix standard samples To set up the run sheet for automatic data analysis Step Action 1 Verify that the data analysis software is selected in Preferences To do this a Open the Window menu and select Preferences b Select GeneScan Run Defaults c Look at the Autoanalyze with field Preferences gt Page GeneScan Run Defaults v Operator wIR 12 w em Lanes 24 v PreRun Mnie Run medue mone Analysis software not selected E Autoanalyze with Analysis Parameters _ lt Analysis Default w Gal s Matrix File Size Standard Auto Print Co If the analysis software is Then selected click OK to return to the run sheet not selected a Open the Autoanalyze with pop up menu and select Other b In the dialog box locate the ABI PRISM DNA Sequencing Analysis Software and click Open c Close the current run sheet and create a new run sheet Note Changing the preference has no affect on run sheets created prior to the change Verify that the Auto Analyze boxes are selected for each non matrix standard sample Figure 9 1 on page 9
375. sary launch the data collection software Instructions are listed under Launching the Data Collection Software Program on page 9 8 Open the File menu and select New Click the icon named GeneScan Sample Entering To import sample names from tab delimited text files follow the instructions listed Sample Names under Importing and Exporting Sample Sheet Information on page 9 27 Otherwise follow this procedure To enter sample names Step Action 1 Enter sample names in the Sample Name column by clicking in the appropriate Sample Name field and typing the sample name Enter names in the exact order the samples will be loaded onto the gel The numbers to the left of the Sample Name column represent the gel lane numbers Leave fields blank that correspond to empty lanes IMPORTANT Each sample must have a unique name Limit sample names to 27 characters including the default characters Do not use colons slashes or symbols in sample names Note More text can be entered than is visible Text automatically shifts as the information is entered Use the keyboard arrow keys to scroll through long entries 9 22 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To enter sample names continued Step Action 2 If matrix standard samples are being run enter a name for each matrix sample T
376. scence emitted by an ABI PRISM dye will fall within a small wavelength detection range some fluorescence emission in the detection ranges of the other dyes will always occur The multicomponent matrix compensates for this overlap by subtracting out in each dye s detection range the portion of the signal due to fluorescence from other dyes Why Matrices Must Be Remade Factors Affecting When creating a matrix each relevant dye matrix standard must be run separately to Matrix Quality determine the proportional amount of fluorescence that is emitted in all four detection regions Because the emission spectra of the dyes vary with the physical environment such as the pH or polymer type and concentration the matrix must be remade if run conditions change Factors that affect matrix quality are Aging reagents Buffer type and concentration Gel polymer type Denaturing vs non denaturing conditions Run temperature gt gt ad Change in instrument optics CD camera or lenses Virtual Filter Set C The emission maximum of 6 FAM the recommended blue displaying dye for this filter set is very close to the laser wavelength of 514 5 nm Thus the window for collected blue light intensity data is offset to longer wavelengths and does not contain the emission maximum of 6 FAM It is also very close to the detection region for the green displaying dye TET Matrix files made for Virtual Filter Set C are especially susceptible to mino
377. selected proceed to step 6 More Choices is selected a Click the Apple RGB icon to display the window shown below b Use the bars or the fields to create a new color c When finished choose one of the following Click OK to save the change and return to the Dye Indicators preference window Click Cancel to cancel the change and return to the Dye Indicators preference window Click the Apple HSL icon to return to the previous window Apple HSL as Original G O O O Blue EE 62 Fewer Choices To revert back to the factory settings click the Reset to Factory Settings button If Then you are finished click OK to save your changes or Cancel to quit without making any changes you wish to change more preferences open the Page pop up menu and select another Preference Setting Preferences 5 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Project Information Preferences About Project Info This preference is for BioLIMS users only sequencing applications BioLIMS Preferences version 1 0 and up GeneScan applications BioLIMS version 2 0 and up The project information is used to identify the data transferred to BioLIMS a nd must be defined prior to setting up sample sheets If this information is not specified
378. sented in this chapter are Method 1 Using Mounted Plates and the Gel Injection Device Method 2 Using Unmounted Plates Using Mounted Plates and the Gel Injection Device In this method the glass plates are mounted in the gel cassette The gel injection device is attached to the plates and the gel solution is injected between the plates using a syringe Instructions for this method are present in three parts Method 1 Part 1 Mounting Glass Plates into the Gel Cassette on page 2 10 Method 1 Part 2 Attaching the Gel Injection Device on page 2 14 Method 1 Part 3 Pouring the Gel Using Mounted Plates on page 2 18 The advantages of using this method are The gel cassette Eliminates the need for binder clips Ensures the proper amount of pressure is applied to the plates at the correct locations along the edges of the plates Gel preparation is more consistent Using Unmounted Plates In this method the glass plates are clamped together with binder clips and the gel solution is manually injected between the plates Instructions for this method are listed under Method 2 Pouring the Gel Using Unmounted Plates on page 2 20 A summary of the steps involved in preparing a gel are as follows Step Action 1 Clean the glass plates spacers and comb 2 Mount the plates and spacers in the gel cassette and attach the gel injection device method 1 only or Assemble
379. separation due to insufficient heat denaturation Make sure the samples are heated at 95 C for 5 minutes prior to loading in gel Misshapen wells Note If only a few wells are misshapen you can still load the gel using only those lanes with flat well formed wells To prevent suction when removing the comb a Lay gel flat b Pour 1X TBE over comb c Remove comb slowly Ensure that no air is trapped by comb during insertion Wrong TBE buffer formulation Remake buffer carefully following protocol Catastrophic loss of resolution bands tilted and poorly resolved Gel extruding from between plates See Gel Extrusion on page 4 29 for more information Clean the plates with an alcoholic KOH wash page 4 30 or a 3 M HCI wash page 4 31 Troubleshooting 4 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Observation Possible Causes Recommended Actions Poor resolution Poor quality reagents especially acrylamide Use fresh reagents from a reliable source Small bubble s between load and read region Clean plates thoroughly Cast gel carefully Remove bubbles by tapping plates while pouring See Chapter 2 for more information Well shape not flat Check for air bubbles trapped by comb Remove comb carefully Only load in flat wells gt gt Do not push shark s tooth comb
380. sis voltage gel temperature etc required for instrument operation the plate check prerun and run ABI PRism Data Collection Software version 2 1 modules can be grouped into two general categories Standard modules Provide gel temperature control from 10 C above ambient to a maximum of 60 C Located in the Modules folder Chiller modules Modules used when an external cold water bath is attached to the instrument and temperatures below 10 C above ambient are required Located in the Chiller Modules folder See Chiller Modules on page 9 44 for more information Within each of these categories are prerun and run modules specific for sequencing or GeneScan analysis applications The application is designated by the module name see Module Naming Conventions below Choosing a module automatically selects a virtual filter set Virtual filter sets dye sets and the various chemistries available are described under Virtual Filter Sets on page 9 51 and DyeSet Primer Files on page 9 49 If the wrong run module and therefore virtual filter set is chosen for a particular chemistry the data will be poor or useless For sequencing applications bases will be miscalled or the data will be meaningless We recommend running the samples again using the correct run module See page 9 47 for a list of ABI PRISM Data Collection Software version 2 1 modules Module Naming The naming conventions used for module fi
381. sistently increase read lengths on 36 cm runs at 2400 scans hr or 48 cm gel runs For 48 cm gel runs 5 25 PAGE PLUS and 4 75 Long Ranger Singel gels give longer read lengths than 19 1 polyacrylamide gels For 36 cm gel runs at 2400 scans hr use a 4 5 29 1 polyacrylamide gel Ingredients and Run For 48 cm well to read runs 4 75 Long Ranger Singel Conditions Ingredients Run Conditions As supplied by the manufacturer Use 48 cm run module Increase run time to 11 hr For 36 cm well to read runs 5 0 Long Ranger Singel Ingredients Run Conditions As supplied by the manufacturer Use standard 36 cm run modules with a run speed of 1200 scans hr Increase run time to 9 hr For 36 cm well to read runs 5 0 Long Ranger 6 M Urea Gel Ingredient For 50 mL Run Conditions urea 18 0g Use standard 36 cm run modules with a run 50 gel stock solution 5 0 mL speed of 1200 scans hr 10X TBE 5 0 mL Increase run time to 9 hr deionized water to 50 0 mL 10 APS 250 uL TEMED 25 0 pL 10 Ammonium IMPORTANT Use fresh ammonium persulfate Prepare the 10 ammonium persulfate Persulfate solution no more than two hours before pouring the gel Crystals should crackle as dissolved Step Action 1 Weigh out 0 50 0 005 g of ammonium persulfate into a 15 mL polypropylene tube WARNING CHEMICAL HAZARD Always wear appropriate safety
382. six cassette clamps that hold the plate in place and lifting it away from the instrument IMPORTANT Always remove the front heat transfer plate from the gel cassette before removing the cassette from the instrument The front heat transfer plate is heavy and removing them together can damage the cassette Siphon the buffer from the upper and lower buffer chambers into an appropriate waste container WARNING Tris borate EDTA TBE buffer can be harmful if inhaled ingested or absorbed through the skin It is irritating to the eyes skin and mucous membranes Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves Open the four clamps that hold the cassette in place and remove the cassette from the instrument Holding the cassette over a sink carefully open the cassette clamps holding the upper buffer chamber to the cassette and remove the buffer chamber Remove the gel plates from the cassette 10 Clean the cassette with damp Kimwipes and allow it to air dry it 11 Remove the lower buffer chamber and discard any remaining buffer 12 Clean up any liquid left in the electrophoresis chamber 13 Clean the front and rear heat transfer plates and the positioning pins with damp Kimwipes Place the front heat transfer plate on a non scratch surface Allow all the parts to air dry 14 Rinse the buffer chambers with deionized water and allo
383. so save the sample sheet by opening the File menu and selecting Save Save As or Save A Copy In Proceed to Preparing a Run Sheet on page 3 28 to finish setting up the software Instrument Operation 3 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Preparing a GeneScan Sample Sheet IMPORTANT Do not mix GeneScan and sequence analysis samples on the same sample sheet or in the same run Save Time by If the same type of run is performed repeatedly on the same instrument you can Setting Sample reduce the time spent setting up sample sheets by setting GeneScan sample sheet Sheet Preferences default preferences Setting sample sheet preferences means changing the default value of certain fields on the sample sheet template to the values used most often The following field can be set as a preference Std size standard dye color Once you have set this preference the preferred value appears automatically on each new GeneScan sample sheet This preference can be changed as often as necessary either by setting a new preference value or by manually selecting a different value on the sample sheet Instructions for setting GeneScan sample sheet default preferences are located in Chapter 5 Setting Preferences Open a New Sample To open a new GeneScan sample sheet and enter sample names Sheet and Enter Sample Names Step Action 1 Open the F
384. spiratory protection suitable for acid vapor When diluting nitric acid ALWAYS ADD ACID TO WATER or to aqueous solutions Obtain a copy of the MSDS from the manufacturer Wear full protective clothing including eye and face coverings rubber gloves and rubber apron WARNING HAZARDOUS WASTE EXPLOSION HAZARD Nitric acid waste must be emptied into the AQUEOUS waste bottle DO NOT mix nitric acid waste with ORGANIC waste products such as phenol The mixture is potentially highly explosive Always work in a fume hood Wear appropriate eyewear clothing and gloves when handling TEMED solutions Dispose of the contents of the waste tray and waste bottle in accordance with all applicable local state and federal health and environmental regulations WARNING ELECTRICAL SHOCK HAZARD The ABI Prism 377 contains a high voltage power supply Although the instrument has been designed with safety features in the door to disconnect the power supply when the door is open please follow procedures as prescribed As with any electrophoresis apparatus be careful during instrument operation and when handling electrodes and liquids Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Procedure To flush the system with a cleaning solution Step Action 1 Power on the instrument Open the panel on the right side of the instrument to access the water reservoir Place paper towels u
385. ssette 3 45 setting folder location preferences 3 51 to 3 53 setting up the software 3 19 skipping lanes determining which to skip 3 14 starting and monitoring the run 3 40 to 3 43 viewing the Log file 3 44 summary of operation procedures 3 2 ABI Prism DNA Sequencing Software run sheets setting preferences 3 28 sample sheets setting preferences 3 19 analysis parameters 3 28 3 33 3 34 3 51 3 52 analyzing data 3 46 See also automatic data analysis arcing instrument damage warning 3 9 why it occurs 3 9 automatic data analysis analysis parameters 3 28 3 33 3 34 3 51 3 52 automatically printing results 3 32 3 33 deselecting 3 31 for GeneScan runs 3 33 for sequencing runs 3 32 B BioLIMS project info preferences 3 19 3 25 projectnames 3 19 3 25 buffer chamber cleaning afterarun 3 45 emptying 3 45 filling procedure 3 16 to 3 17 fixing leaks in upper chamber 3 17 installing lower buffer chamber 3 8 to 3 10 installing upper buffer chamber 3 15 solution levels that must be maintained 3 17 buffer solution levels that must be maintained in buffer chambers 3 17 preparing 1X TBE 3 3 C cables See electrode cables calibration file make function description of 3 49 calibration file send function description of 3 49 cassette See gel cassette CCD Gain function description of 3 49 CCD offset function description of 3 50 CCD pixel position value 3 49 chiller modules folder location and description of 3 51 c
386. t Run Sheet Step Action 1 If necessary launch the data collection software Instructions are listed under Launching the Data Collection Software Program on page 9 8 Open the File menu and select New Click either the Sequence Run or GeneScan Run icon as appropriate A new Run folder is created automatically in the Runs folder inside the ABI PRISM 377 folder Selecting the Plate To select the plate check prerun and run modules Check PreRun and Run Modules Step Action 1 Open the Plate Check Module pop up menu and select the a plate check module b gt KUN Sbr Z4uu Plate Check Module PAE Cia Plate Check B Plate Check C Run Modute piste Check D IMPORTANT If the pop up menus list lt none gt only software cannot find the folder that contains the modules See Setting Folder Location Preferences on page 9 5 Open the PreRun Module pop up menu and select a prerun module Open the Run Module pop up menu and select a run module Artisan Technology Group Data Collection Software 9 31 Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Selecting the To select the number of lanes Number of Lanes Step Action 1 Open the lanes pop up menu and select the appropriate value ee For GeneScan applications For Sequencing applications Full Scan 24 34 36 lanes Full Scan 24
387. t Alerts you to when the disk is being accessed Use the following utilities to improve the performance of your Macintosh Speed Disk Optimizes defragments files on the hard disk to improve computer performance System Info Rates the performance of your Macintosh Fast Find Searches disks to find files by name or category Floppier Makes exact copies of your disks that can be copied to new disks or stored to disk as image files Refer to the Norton Utilities user s manual for more detailed information on this product 8 22 System Maintenance Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Archiving Data from Runs Recommendations Files Created During a Run Run Files Gel Files Log Files Sample files Seq files Other Files For optimal computer performance we recommend the following Keep as few files as possible on the hard disk Do not store gel files and run folders on the hard disk Copy all files you want to save onto a backup medium before each run to ensure adequate disk space is always available When you set up a run the data collection software creates a Run folder and a run file During the run the program automatically creates and saves two files the Gel file and the Log file After the run the analysis program creates Sample and sequence Seq files Each run file contains information that associates specific sa
388. t Primer Files 9 49 Virtual Filter Sets 9 51 Viewing Data and Instrument Status 9 53 Opening and Saving Files 9 57 Archiving Files 9 58 Printing Files 9 58 Quitting the Data Collection Program 9 59 Data Analysis Overview 9 60 Data Collection Software 9 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com System Software Overview Overview 9 2 Data Collection Software The ABI PRism 377 DNA Sequencer is shipped with two types of software ABI Prism Data Collection Software One of the following data analysis software programs ABI PRISM DNA Sequencing Analysis Software ABI Prism GeneScan Analysis Software This chapter describes the ABI PRISM Data Collection Software This software Controls the instrument by sending it commands that are contained in files called modules Collects data from the instrument and stores it in a gel file Transfers data to either sequencing or GeneScan software for analysis The data collection software is installed on the Macintosh computer hard drive during system installation For more information on the data analysis software programs and the chemistries available for use with this system refer to the following manuals ABI PRISM GeneScan Analysis Software User s Manual GeneScan Reference Guide ABI 373 and ABI PRISM 377 DNA Sequencers P N 4303188 ABI PRiISM DNA Sequencing Software User s Manua
389. t Sample A Project 2 Sample B DP4 Ac M1 3Rev PJ Setup Matrix Project 2 Comments about Sample B Project 2 DP4 AcIM1 3Rev P Setup Matrixp Project 2 Is Figure 3 3 Example of a completed sequencing sample sheet Sample Name DyeSet Primer Comments 3 22 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Save and Close the To save and close the sample sheet Sample Sheet Step Action 1 Do one of the following Open the File menu and select Close Click the box in the upper left hand corner of the window and then click Save A dialog box showing the default sample sheet file name and the location where the sample sheet will be stored is displayed a Change the file name now if desired b Click Save IMPORTANT Although it is an option we do not recommend changing the storage location of the sample sheet If the location is changed software will not be able to locate the sample sheet when you set up the run sheet Folder where sample sheets are stored amp Sample Sheets v amp Macintosh HD fu Sample Sheet 5 13 98 3 49 PM Eject fu Sample Sheet 5 13 98 4 20 PM fi Sample Sheet 5 14 98 10 47 Desktop jul Sample Sheet 8 New C Pu Sample Sheet 5 15 Save this document as Cancel i Sample Sheet 5 17 98 11 47 A Sample sheet file name Note You can al
390. t of the plates may cause the bar to break resulting in broken glass plates See the illustration on page 3 4 To load the gel into the cassette Step Action 1 Place the cassette on a clean level surface 2 Orient the plates in the cassette as shown below so the front plate is on top Then push the plates to the bottom of the cassette until the notches in the rear plate are seated firmly against the metal plate stops in the cassette Push the plates from the top to ensure firm contact 3 Press on the center of the front plate with the fingertips of one hand to hold plates in place At the same time lock the plates into position by turning all the cassette clamps to the closed position except for the bottom pair of clamps 4 Lower the laser beam safety bar and turn the bottom cassette clamps to lock the bar into position Laser beam safety bar lowered and locked in place by these two clamps Plate stops 5 If using a Then shark s tooth comb a make sure the comb is clean and dry b Proceed to the next step square tooth comb proceed to Installing the Gel Cassette and Lower Buffer Chamber on page 3 8 6 If this is Then not a 96 lane upgrade proceed to the next step instrument a 96 lane upgrade using a syringe add 1X TBE buffer to the loading area instrument Adding TBE makes comb ins
391. t peaks are present Open the File menu and select Quit to quit the Sequencing Analysis program Make Backup The sample files used to make an instrument file are altered by the DataUtility Copies of Raw Data program when the instrument file is generated Therefore it is important to make backup copies of the standard sample files the raw unanalyzed data before making the instrument file If the new instrument file is no good you can create another one using the backup copies of the sample files Making Instrument Files for Sequencing 7 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Making the At this point in the procedure you should have run the necessary matrix standard Instrument File samples Running Matrix Standards to Obtain the Raw Data on page 7 6 generated sample files and verified the raw data Verifying the Raw Data on page 7 7 and made backup copies of the sample files To make an instrument file for virtual filter set A Step Action 1 Open the DataUtility program This program is located in the Utilities folder inside the Sequencing Analysis folder 2 Open the Utilities menu and select Make Matrix The Make Matrix dialog box appears Make Matrix Name of file containing C data Start at Name of file containing A data Start at 6 Name of file containing G data Start at Name of file containing T data Start
392. t the entire column open the Edit menu and select Fill Down to enter the same instrument file for the remaining samples Instrument Operation 3 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Select Project Names The Project Name field is for BioLIMS users only Otherwise the field can be left and Enter blank If a project name is not identified the data transferred to BioLIMS is identified Comments by the gel file name only Project names must be defined prior to setting up the sample sheet as Project Info Preferences Refer to Project Information Preferences in Chapter 5 for instructions and more information To select project names and enter comments Step Action 1 Open the Project Name pop up menu for each sample and select the appropriate project name Project Name Project 1 Project 2 Project 3 2 If the Project Name is the same for all remaining samples click in the column heading to select the entire column open the Edit menu and select Fill Down Otherwise select the appropriate Project Name for each sample individually 3 To enter comments click in the Comments field and type the information Sample Sheet Sample Sheet 5 19 98 11 01 AM Sequencing Sample Sheet Instrument Project Name File DP4 Ac M1 SRevt Ese metro p gt Project 2 P Comments abou
393. t transfer plate installing 3 18 removing and cleaning 3 45 G gel cassette cleaning afterarun 3 45 electrical shock hazard warning 3 9 how to carry with glass plates 3 4 installation procedure 3 8 to 3 10 loading gel plates into cassette 3 6 to 3 7 removing from instrument 3 45 gel extrusion preventing 3 35 gel temperature determining 3 36 viewing in Status window 3 36 Gel window description of 3 42 gels removing from glass plates 3 45 GeneScan analysis parameters folder location and description 3 52 GeneScan size standard folder location and description 3 52 glass plates cleaning afterarun 3 45 cleaning before loading gel 3 4 to 3 5 read region description of 3 47 Index 2 heat transfer plate instrument operation H laser standby function description hard disk of 3 49 load volumes suggested 3 39 loading samples how to 3 35 skipping lanes 3 38 loading solution preparing loading solution 3 3 using formamide in empty wells 3 38 ensuring space for automatically created files 3 40 rear cleaning afterarun 3 45 See also front heat transfer plate I Log file cleaning glass plates 3 4 to 3 5 description of and viewing 3 44 cleaning up after run 3 45 Log window See Log file filling buffer chambers 3 16 to lower buffer chamber TO filling 3 16 installing front heat installing 3 8 to 3 10 transfer plate 3 18 installing gel cassette 3 8 to 3 10 installing lower buffer M chamber 3 8 to 3 10 ma
394. te check electrical shock hazard warning 3 11 how to perform 3 11 plates cleaning before loading gel to 3 5 read region description of positioning pins checking cassette installation 3 10 cleaning afterarun 3 45 power switch 3 8 preferences for GeneScan sample sheets 3 24 forrun sheets 3 28 for sequencing sample sheets 3 19 Project Info preference 3 19 3 25 setting folder locations 3 51 to 3 53 3 4 3 47 prerun performing prerun and loading samples 3 35 to 3 39 printing data from runs 3 46 project names how to define 3 25 purpose of 3 25 pump off function description of pump on function description of 3 49 3 49 R read region description of run cleaning up after run 3 45 starting and monitoring 3 40 to 3 43 run sheets how to prepare 3 28 to 3 34 run temperature determining 3 36 viewing in Status window 3 36 run window Same as run sheet runs folder location and description of 3 51 3 47 S U sample files upper buffer chamber location of 3 51 See also buffer chamber sample loading filling 3 16 suggested load volumes 3 39 installing 3 15 sample names restrictions 3 20 3 24 WwW sample sheet folder location and Wells description or 14 51 flushing before loading sample sheets samples 3 35 changing after selecting on run using formamide in empty sheet 3 30 wells 3 38 preparing for GeneScan run 3 24 preparing for sequencing run 3 19 to 3 23 samples loading ergonomic warning and sugges
395. tem File 8 29 System Maintenance 8 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Instrument Maintenance Recommendations How to Maintain ELECTRICAL HAZARD The ABI Prism 377 contains a high voltage power supply the Instrument Although the instrument has been designed with safety features in the door to disconnect the power supply when the door is open please follow procedures as prescribed As with any electrophoresis apparatus be careful during instrument operation and when handling electrodes and liquids Recommendation For more information see After every run Cleaning Instrument Wipe the front and rear heat transfer plates and Accessories on page 8 3 positioning pins with damp lint free towels and Chapter 2 Pouring Gels allow to air dry Chapter 3 Instrument Remove any liquid present in the electrophoresis Operation chamber Wipe the gel cassette clean with a damp lint free towel and allow to air dry Clean the comb and spacers with deionized water Do not use an organic solvent Rinse the buffer chambers with deionized water and allow to air dry Weekly Check the water reservoir level and refill as Refilling the Water Reservoir on necessary page 8 5 8 2 System Maintenance Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg co
396. tenance Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Computer Maintenance Recommendations How To Maintain Maximum Operating Efficiency Computers require regular maintenance to operate efficiently and consistently Because the ABI PRISM 377 software works with large files and accesses the hard disk often it is important to follow the procedures described in this section Adherence to these recommendations will minimize the occurrence of errors during operation and will help maintain maximum operating efficiency Recommendation For more information see Restart the Macintosh before each run Restarting the Computer on page 8 18 Rebuild the desktop Once a month After installing new software After running Norton Utilities Rebuilding the Desktop on page 8 18 Delete or store on another medium and then delete data files and other non essential files from the hard disk at least once a week Data files are gel files sample sheets run sheets and sample files This practice will ensure that enough disk space is always available to run the instrument Because gel files are so large 20 70 MB we recommend deleting them as soon as satisfactory sample files are generated Deleting Data Files on page 8 18 Archiving Data from Runs on page 8 23 Includes a short description of each type of data fi
397. tent reagent purity and associated problems 3 5 inhibited by buffer impurity 3 6 Mutation Detection gel See MDE gel 3 8 air bubbles 3 8 TEMED 3 6 rate of polymerization 3 7 poor resolution caused by acrylamide reagent purity 3 5 to 3 6 impurity 3 5 preparing 3 2 porous gel factor that affects rate of polymerization 3 7 protocols chemical abbreviates used 3 9 Index 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com factors that affect read lengths 3 4 list of recipes and recommendations 3 2 Long Ranger gels 10 ammonium persulfate 3 15 4 75 Long Ranger Singel 3 16 5 Long Ranger gel for SSCP 3 17 not for SSCP 3 16 to 3 17 ingredients and run conditions 3 15 MDE gel protocol 3 18 PAGE PLUS gels ingredients and run conditions 3 13 protocol 3 14 Polyacrylamide gels gel solution protocols 3 19 to 3 21 ingredients and run conditions 3 11 protocol 3 11 to 3 12 sequencing chemistries 3 3 purity effect of urea impurity 3 6 Q quality temperature and gel quality 3 8 R read lengths factors that affect reagents purity how it affects gel quality 3 5 to 3 6 storing 3 23 reproducibility affects of acrylamide impurity 3 5 buffer impurity 3 6 vacuum strength and time during degassing 3 7 3 4 resolution affects of acrylamide impurity 3 5 deterioration due to aging gel 3 8 Index 2 Artisan Technology Group Quality Instrumentation Guarant
398. tes are even slightly misaligned the gel injection device will leak Bottoms of plates Misalignment like this causes correctly aligned the gel injection device to leak 2 12 Pouring Gels Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To mount glass plates in the gel cassette continued Step Action 9 Press on the center of the front plate with the fingertips of one hand to hold the plates in position At the same time lock the plates in place by turning all the cassette clamps to the closed position except the clamps that secure the top and bottom of the plates shown below SS pee E o oaeo These clamps are left open for 36 cm glass plates 10 Run the tip of your finger along the bottom of the plates again to make sure they are still flush If the plates shifted unlock the clamps and adjust the plates Repeat this procedure until the plates are locked in position against the plate stops and the bottoms of the plates are flush Pouring Gels 2 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Method 1 Part 2 Attaching the Gel Injection Device Materials Required The materials required for this procedure are as follows Gel injection device three pieces as illustrated below Gel injection fixture clear Two braces that hold the
399. the manufacturer Wear appropriate protective eyewear clothing and gloves 2 Add 200 mL of absolute ethanol to the bottle WARNING CHEMICAL HAZARD Ethanol is a flammable chemical and is irritating to the skin eyes respiratory system It can cause nerve and liver damage CNS depression nausea vomiting and headache Always work in a fume hood Obtain a copy of the MSDS from the manufacturer Wear appropriate protective eyewear clothing and gloves 3 Mix the solution well It will take at least 15 minutes for most of the pellets to dissolve Note This recipe is for a saturated solution so some pellets will remain Store the solution with the bottle capped tightly During storage the color of the solution will turn dark red brown The solution can still be used and is good for 1 year 4 Place some uncolored absorbent towels or other covering in the hood to catch Spills 5 Place the gel plates on the towels with the inside surfaces facing up Note The plates should be nearly level so that the cleaning solution does not run off onto the bench Only the inside gel side surface of the plates need be cleaned though the outside surfaces can be cleaned similarly 6 Pour approximately 15 mL of the cleaning solution onto the center of each plate to be cleaned Spread the solution over the surface of plate 7 Allow the solution to remain on the plates for 5 minutes CAUTION Longer times can harm the plates
400. the Manual Control window and try to turn the pump on manually If the problem persists call service Error message No Flow Detected Attempted Pump Restart Coolant pump was turned on but no coolant flow was detected by the flow switch Check the water reservoir Refill the bottle if necessary Procedure listed in Chapter 8 System Maintenance Error message Scanner Did Not Find Its Home Position Scanner did not find home position prior to collecting data for a plate check prerun or run Press the Reset button on the back of the instrument once Then click Resume in the run window Error message A Valid XL Lane Firmware Image is Required Non XL data collection software tried to establish communication with an ABI PRISM 377 instrument with XL upgrade Install and use ABI PRISM 377 XL Upgrade Data Collection Software Error message A Valid 96 Lane Firmware Image is Required Non 96 data collection software tried to establish communication with an ABI PRISM 377 instrument with the 96 lane upgrade Install and use ABI PRISM 377 96 Lane Upgrade Data Collection Software Error message Heat plate temperature exceeds 70 C even when status window indicates less than 70 C Clog in coolant system Flush the coolant system to remove the clog and refill the water reservoir with deionized water and 5 0 antifreeze See Removing Coolant System Clogs
401. the four regions These appear as the blue green black yellow on gel images and red peaks in the raw data This process is similar to using a physical filter to separate light of different wavelengths However the filter sets are virtual because the instrument uses no physical filtering hardware to perform the separation Each virtual filter set has been optimized to provide the maximum possible separation among the centers of detection for the different dyes used together as a set while maintaining an excellent signal to noise ratio The virtual filter used during data collection is designated by the run module selected on the run sheet The exact positions of the CCD regions and the dye combinations appropriate to these positions depend upon the virtual filter set used For example with Virtual Filter Set E the instrument records the light intensity in four regions or windows centered at 540 nm 570 nm 595 nm and 625 nm The window positions in each virtual filter set have been optimized to provide the maximum possible separation among the centers of detection for the different dyes while maintaining good signal strength The data collection software color codes the intensity displays from the four light collection regions These appear as the blue green black yellow on gel images and red peaks in the raw data The Sequencing Analysis Software uses the same four colors to color code analyzed data from all dye virtual f
402. the plates and spacers using binder clips method 2 only Prepare the gel solution Add the polymerizing reagents to the gel solution Pour the gel Insert the comb Wrap bottom of plates with plastic wrap O NIO O Allow the gel to polymerize Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Before Using New Glass Plates The Gasket Mark Identifying the Front and Back of a Plate The first time the front glass plate and the gasket of the upper buffer chamber make contact an invisible permanent hydrophobic area is created on the surface of the glass This hydrophobic area is referred to as the gasket mark If subsequent gels are poured using the same plates with the gasket mark on the inside the gel solution will not flow readily across this area of the plate Therefore always keep the same side of each plate facing out Gasket marks can be removed by cleaning the plates with an alcoholic KOH wash See Removing the Gasket Mark on page 2 9 The sides of glass plates can be identified several different ways Applied Biosystems glass plates are scored with the part number and well to read distance You can orient plates so these identifiers always face out In addition you can Number the front and back plates as a set and always use the same two plates together The benefit of this method is that gel related problems can m
403. the same instrument Using the same run modules set of dyes gel parameters gel polymer type buffer denaturing or non denaturing conditions etc After running the matrix standards use their sample files to generate a matrix file using ABI PRISM GeneScan Analysis Software Verify the Raw Data Before creating the matrix file verify that the raw data from the standards is good To view the raw data in GeneScan analysis software Step Action 1 Create a new project if you did not select Autoanalyze in the GeneScan Run Defaults preferences in the data collection software a Choose New from the File menu Select the Project icon An untitled Analysis Control window opens Choose Add Sample Files from the Project menu Find and open the Run Folder for the matrix standards run ooo F Select the four Sample files representing the blue green yellow and red dye labeled runs and then click Add f Click Done after the Sample files are transferred 2 In the Analysis Control window select the four matrix standard Sample files by clicking on the first Sample file holding down the mouse button and releasing on the last Sample file 3 Choose Raw Data from the Project menu Electropherograms displaying raw data from the four matrix standard Sample files appear To verify the raw data Step Action 1 Verify data peaks are present in all four samples Peak data should
404. the same type of run is performed repeatedly on the same instrument you can reduce the time spent setting up sequencing sample sheets by setting sequencing sample sheet default preferences See Save Time by Setting Sample Sheet Preferences on page 9 16 and Chapter 5 Setting Preferences for more information An instrument file is specified on both the sample and run sheets The same file should be specified on both sheets However if different files are selected the instrument file specified on the sample sheet is the one used for automatic data analysis If lt none gt is selected on the sample sheet the instrument file selected on the run sheet is used Information can be imported from tab delimited text files for instance files generated by a database The sample sheet can be exported as a tab delimited text file to database spreadsheet or word processing programs Refer to Importing and Exporting Sample Sheet Information on page 9 27 for more information The same sample sheet can be used for more than one run if the information required is the same An existing sample sheet can also be used as a template to create new sample sheets by opening the file saving it under a different name Save As command under the File menu and then modifying the new file If the wrong dye set primer or instrument file is specified the data can be reanalyzed using the correct file Refer to the AB Prism DNA Sequencing Anal
405. tifies multiple base positions with codes described by the International Union of Biochemists IUB codes based on a user defined threshold The program processes sequences in batches speeding the process considerably AutoAssembler allows you to quickly and efficiently assemble small pieces of DNA into larger segments of DNA using ABI Prism 377 data as well as other types of data It provides powerful tools for editing the sequences including the ability to display constantly spaced electropherograms that are synchronized with the assembled sequences You can build a consensus from the assembled sequences and export it for use with other programs GeneAssist Analysis allows you to analyze data from ABI Prism 377 sequence files files processed in AutoAssembler text files or a database You can use this program to rapidly search biological databases for specific sequences or motifs and to interactively work with the resulting sequence lists In addition to sequence lists the program can create dot plots alignments and restriction maps of sequence data AutoAssembler simplifies sequence assembly by automating each step of the process from vector deactivation sequence cleanup and assembly to easy resolution of sequence ambiguities It is designed for use on small to medium size sequencing projects AutoAssembler automatically examines all possible relationships between sequences in both orientations Dynamic programming i
406. tinue Select the text box and type the correct CCD pixel position value Click Execute Entering the CCD Pixel Position Value Step 1 2 3 5 6 If the CCD Pixel Position error occurs again call Applied Biosystems technical support telephone numbers listed in Chapter 1 4 26 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Using Calibration File Make and Send Function Description Using Calibration File Make Two functions Calibration File Make and Calibration File Send allow you to create a file that contains the CCD pixel position value and instrument serial number This file is called ABI 377 Calibrations and is stored in the Preferences folder inside the System folder The benefits to using these functions are as follows Simply use Calibration File Send to reenter the CCD pixel position value rather than manually locating and reentering the value as described under CCD Pixel Position Value on page 4 25 Once Calibration File Send is executed the instrument serial number is added to all sample files and is visible when sample files are opened in the annotative view This is useful for tracking and troubleshooting particularly when more than one instrument is in operation IMPORTANT The correct CCD pixel position value must be entered in instrument memory before using Calibration File Make See Locat
407. tion Try the following Wash glass plates in laboratory dishwasher with hot deionized water rinse cycle 195 F 90 C Soak plates overnight in a 5 0 solution Multiterge See Chapter 2 for more information on cleaning glass plates Inconsistent signal from lane to lane Some samples not thoroughly mixed with formamide size standard mixture Mix samples into formamide size standard mixture by pipetting up and down several times Signal too low peak heights lower than usual Insufficient sample added to some or all lanes Cassette not flush with back heat transfer plate and alignment pins Check your protocol Examine the efficiency of the PCR Check pipette calibration Refer to the GeneScan or Sequencing guides listed on page 4 2 for more information Place cassette flush against back heat transfer plate IMPORTANT The spacers must touch the alignment pins You should be able to see the alignment pins pressing against the spacers Difficulty in loading sample because wells not flushed Flush the wells prior to loading the gel Bad formamide Use freshly deionized formamide Procedure in Appendix A Note Formamide pH should be between 7 and 9 Insufficient FJdNTPs added to PCR reaction Reamplify using more F dNTPs or examine the efficiency of the PCR Optics detector misaligned Call service representative Troubleshooting 4 11 Artisan
408. tion 194 gt 2 20 98 5 04 09 PM 377 Controller board Rev Bor later 10 MHz 30K The gt 2 20 98 5 04 09 PM Scan Line Averaging On 2 20 98 5 04 40 PM Message Starting scan The information in the file is formatted as follows XXX mm dd yy hh mm ssdescription The entry in the first column xxx is variable Possible entries are sas information system start or stop file created gt message sent to instrument lt message received from instrument lal warning HHH error The second column shows the month day year and hours minutes seconds The third column is a brief description of the event 9 54 Data Collection Software Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The Scan Window The Scan window below shows real time raw data as sweeps of the laser across the gel The different colored lines represent each dye color in the dye set Data is updated every few seconds To display the Scan window open the Window menu and choose Scan To set the scale of the Scan window open the Edit menu and choose Set Scale Enter minimum and maximum values for the scale in the dialog box that is displayed E Sean Window SEEE x 191 Y 4783 81924 Freeze Updates Current Scan Number 1088 6144 4096 4 20484 PPA The Gel Window The Gel window is a reconstruction of actual data The first fragments passing the
409. tions 3 37 performing prerun and loading samples 3 35 to 3 39 saving automatically created files ensuring disk space 3 40 Scan window description of 3 41 use during Plate Check 3 11 sequencing software automatic data analysis 3 32 settings ABI folder location and description of 3 52 settings changing manually 3 48 shark s tooth comb inserting 3 6 skipping lanes determining which to skip 3 14 software setting up for a run 3 19 staining wells 3 35 status lights on instrument 3 8 Status window description of 3 42 viewing gel and run temperatures 3 36 stop and analyze feature using 3 40 T temperature run 3 35 Tris borate EDTA chemical warning 3 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Index 3 rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com
410. to products or parts furnished by third parties THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND IS IN LIEU OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS Applied Biosystems Limited Warranty D 1 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Index Numerics 10X TBE buffer solution 3 2 3 3 A 6 A 9 1X TBE buffer solution preparing 3 3 A 9 3 M HCI Wash procedure 4 31 377 instrument See ABI Prism 377 377 with 96 lane upgrade 1 12 377 with XL upgrade 1 11 377 18 1 11 A ABI settings folder location and description of 3 52 ABI Prism 377 changing settings in real time 3 48 cold boot procedure 4 23 data analysis overview 1 18 to 1 19 data flow to computer 9 7 to 9 8 description of instrument 1 11 firmware image downloading file 4 24 how the instrument works 1 13 instrument operation an overview 1 14 to 1 17 cleaning the glass plates 3 4 to 3 5 cleaning up after run 3 45 filling buffer chambers 3 16 to 3 17 installing front heat transfer plate 3 18 installing lower buffer chamber 3 9 installing upper buffer chamber 3 15 loading gel into cassette 3 6
411. to 50 mL IMPORTANT Be sure to use disodium EDTA to make 10X TBE stock Some major laboratory suppliers provide monosodium EDTA or tetrasodium EDTA Mix ingredients thoroughly by vortexing Verify that the pH is between 8 2 and 8 3 Note 10X TBE stored at room temperature should be used within 1 month Do not use if a precipitate is present IMPORTANT Discard if the pH is not 8 3 0 2 and make a fresh solution Do not attempt to adjust the pH IMPORTANT Do not use 10X TBE buffer which has precipitated The change in ion concentrate affects sample migration 1X TBE Working solution 1X is 89 mM Tris base 89 mM Boric acid 2 mM Na EDTA pH is approximately 8 3 at ambient temperatures To prepare a 1X TBE working solution Step Action 1 Add 120 mL 10X TBE stock solution to a large graduated cylinder 2 Dilute with deionized water to a total volume of 1200 mL Gel Recipes A 9 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Deionizing Formamide Procedure IMPORTANT Always use deionized formamide Over time formamide hydrolyzes to formic acid and formate Deionized formamide stock lasts for 3 months at 15 to 25 C Step Action 1 Mix 50 mL of formamide and 5 g of AG501 X8 ion exchange resin WARNING CHEMICAL HAZARD Formamide is a teratogen and is harmful by inhalation skin contact and ingestion Use in a well ventilated area Use che
412. to 8 24 instrument files 7 19 recommendations for 8 20 See Also archiving and saving BigDye primers associated filter set 7 4 kit A 3 BigDye terminators associated filter set 7 4 kit A 3 BioLIMS description of 1 22 project info preferences 3 19 3 25 5 20 projectnames 3 19 3 25 bis acrylamide safety warning A 5 A 23 bleedthrough peaks 6 3 buffer chamber cleaning afterarun 3 45 emptying 3 45 filling procedure 3 16 to 3 17 fixing leaks in upper chamber 3 17 installing lower buffer chamber 3 8 to 3 10 installing upper buffer chamber 3 15 replacing upper buffer chamber gasket 8 6 to 8 11 solution levels that must be maintained 3 17 buffer chamber upper gasket kit part numbers C 3 buffer solution how impurities affect reproducibility A 6 levels that must be maintained in buffer chambers 3 17 preparing 10X TBE A 9 preparing 1X TBE 3 3 A 9 C cables See electrode cables calcium buildup removing 4 32 to 4 33 Calibration file Calibration File Make 4 27 to 4 28 Calibration File Send 4 27 to 4 28 See Also CCD camera and CCD pixel position value sending CCD pixel position value to instrument 8 16 calibration file make function description of 3 49 calibration file send function description of 3 49 cassette See gel cassette CAUTION user attention word defined 1 5 CCD camera aligning 4 25 to 4 26 color and virtual filter sets 7 4 pixel position value 8 14 to 8 16 using Calibration File Make 4 27 to 4 28 using
413. to avoid transferring fluorescent contaminants from your hands to the glass plates To clean the glass plates Step Action 1 If the plates are Then very dirty a remove the plates from the cassette to clean them if applicable CAUTION Always remove the plates from the cassette before rinsing in a sink with tap water Arcing can occur inside the instrument if the cassette and plates are installed with even a small amount of moisture on them Arcing is a luminous low voltage high current electrical discharge that can severely damage the instrument b Put the plates in a sink and rinse them with cool tap water making sure the read region ont see page 3 47 is thoroughly cleaned c Dry the plates with Kimwipes 3 4 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To clean the glass plates continued Step Action 1 not very dirty a you can leave them in the cassette to clean cont them if applicable b Using deionized water and Kimwipes thoroughly clean the read region see page 3 47 c Dry the plates with Kimwipes IMPORTANT Be sure to clean the outside of the back plate Gel solution can sometimes leak out the back while pouring the gel and polymerizes as drops on the glass If not removed the back plate will not make full contact with the rear heat trans
414. to be analyzed automatically at the end of a run For more information refer to About Automatic Data Analysis on page 9 38 Chapter 7 Making Instrument Files for Sequencing Automated DNA Sequencing Chemistry Guide P N 4305080 Chemistry kit protocol Lanes Number of lanes in the gel Run Mode This field is displayed for ABI PRISM 377 XL or 96 lane upgrade XL or 96 lane upgrade instruments only It is used to designate the scan mode 96 Lane instruments only Scan XL Scan or Full Scan Operator Operator s name Sample Number These fields are filled in automatically when the Sample Sheet is Sample Name selected Sample File Name Auto Analyze When selected data is automatically transferred to sequencing analysis software and analyzed at the end of a run See About Automatic Data Analysis on page 9 38 for more information Auto Print When selected analyzed data is printed automatically Can be set as Preferences See Setting Run Sheet Preferences on page 9 31 The following is a new GeneScan run sheet for standard ABI PRISM 377 instruments The run sheet for instruments with the XL or 96 lane upgrade differs slightly as noted in the following table a Run 2 6 98 11 07 AM LE Plate Check Module lt none gt PreRun Module Run Module lt none gt vj D Collect time 3 0 hours Sample Sheet lt none gt v D Well to Read distance
415. tomatic data analysis Clicking Stop amp Analyze terminates the run and starts automatic data analysis of the data collected to that point in the run The run cannot be resumed To use this feature the run sheet must be configured for automatic data analysis prior to starting the run Clicking Continue resumes the run and data collection Data Collection Software 9 39 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The following table is an example of a typical series of commands that would be used for a sequencing run This example is written as if a shark s tooth comb is being used and samples are being loaded using a staggered load method Refer to Chapter 3 Instrument Operation for more information on sample load methods and recommendations Button Clicked Module Affected Action Plate Check Plate Check module Starts the plate check Plates are scanned without electrophoresis to check for fluorescent contaminants N A Terminates the plate check Buffer chambers are then filled and checked for leaks Front heat transfer plate mounted if applicable PreRun module Starts the prerun Gel is electrophoresed without data collection to equilibrate gel temperature PreRun module Pauses the prerun Wells flushed and samples loaded into the odd numbered lanes this is the staggered load method Gel temperature is maintained
416. trophoresis Power So w Laser Power mi CCD Offset CCD Gain 0 Saves Default Shown above is the Module Settings dialog box for the module named GS PR 36F 2400 6 Close the run sheet when finished Chiller Modules Chiller modules and an external cold water bath operating at 22 C and below allow you to perform PCR Single stranded Conformation Polymorphism SSCP analysis For more information on using chiller modules and an external cold water bath refer to Appendix B Subambient Temperature Operation For more information on the PCR SSCP technique refer to PCR SSCP Analysis A Guide to Fluorescent PCR Single stranded Conformation Polymorphism Analysis on the ABI PRISM 377 DNA Sequencer P N 904413 Installing Chiller Chiller modules are installed by moving them into the Modules folder with the standard Modules modules or by setting the Folder Locations Preference to allow the software to access them directly in the Chiller Modules folder Moving Chiller Modules to the Modules Folder To move chiller modules to the Modules folder Step Action 1 Double click the hard drive icon on the Macintosh to access the Finder and open the hard drive directory Open the ABI PRISM 377 folder Open both the Modules folder and the Chiller Modules folder Click in the Chiller Modules window so it becomes the active window or OIN Open the Edit menu and choos
417. ty program 7 16 Making Instrument Files for Sequencing Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Using Matrix To verify the instrument file using matrix standards Standards Step Action 1 Open the Sequencing Analysis program 2 Open the matrix standard files that were used to create the instrument file 3 Use the electropherogram analyzed data view to confirm that the analyzed data looks good In each file you should see one color trace with obvious peaks and all other color traces should be flat throughout the run A pattern of pronounced peaks or dips in any of the other three colors indicate that something is wrong If all the data looks good store and backup the new instrument file See Storing and Backing Up the Instrument File on page 7 19 If the data does not look good pick a different range of raw data points and remake the matrix Be sure to use the backup copies of raw unanalyzed data files An analyzed file cannot be used to make an instrument file Refer to the ABI PRISM DNA Sequencing Analysis Software User s Manual for guidelines and a worksheet for selecting new start and data points Making Instrument Files for Sequencing 7 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Making an Instrument File from a Sample File Intr
418. uccessful run Plates that are cleaned thoroughly and consistently will also help avoid the temporary loss of signal that can occur sporadically on this instrument Our research indicates this loss of signal is due to contaminant molecules surfactants fatty acids long chain polymers attached to the surface of the plates It manifests itself as a band of little or no signal across the entire width of the gel image It usually occurs between 140 to 200 base pairs and typically lasts the equivalent of 20 to 40 base pairs Following this band signal strength usually returns to normal Glass plates can be cleaned manually or in a dishwasher The use of a laboratory dishwasher with a hot 195 F 90 C deionized water rinse cycle has been found to effectively remove suspect contaminants thereby eliminating any temporary loss of signal We strongly recommend cleaning glass plates in a laboratory dishwasher with a hot 195 F 90 C deionized water rinse cycle Using a dishwasher helps ensure plates are cleaned effectively and consistently every time and will also eliminate the sporadic temporary loss of signal that can occur on this instrument described above Deionized water is required for the rinse cycle only Dishwasher recommendations are listed on page 8 4 System Maintenance 8 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com When using a dishwasher we recommend you Conn
419. uence and GeneScan analysis samples on the same sample sheet or in the same run Sample Sheet untitled SSS BE Sequencing Sample Sheet Sample Name DyeSet Primer Instrument Project Name Comments File lt none gt lt none gt PJ PJ lt none gt lt none gt PJ Description of Fields The information specified on sequencing sample sheets is described below on the Sample Sheet Name of Field Description Sample Name Name assigned to a sample IMPORTANT Each sample must have a unique name Limit sample names to 27 characters including the default characters Do not use colons slashes or symbols in sample names DyeSet Primer Also referred to as a mobility file Contains the information used to Compensate for differences shifts in sample mobility Interpret what each dye color in a set represents A dye set primer file must be selected for data to be analyzed automatically at the end of a run For more information see About Automatic Data Analysis on page 9 38 DyeSet Primer Files on page 9 49 Project Name Used to identify data transferred to BioLIMS software If a project name for BioLIMS is not specified the data transferred to BioLIMS is identified by the gel file name only software users only Project names must be entered in the Project Info Preference prior to preparing a sample sheet Refer to Project Information Preferences in Ch
420. uit Warning Possible Plate P44 J44 Thermistor Open Short Circuit Warning Plate In Thermistor P44 J44 Open Short Circuit Warning Possible Heater Thermistor Open Short Circuit Possible open or short circuit exists with the thermistor cable connected to J43 or J44 Temperature of the plate in an instrument with 100k ohm thermistors is 21 9 C or less One of two temperature sensors thermistors on the rear heat transfer plate may be bad The instrument will function normally with one sensor Schedule a service call to have the thermistors checked Click OK and continue operating the instrument This message may appear when you launch data collection software and start a plate check prerun or run Error message Flow Detected with Pump Off External Cooling in Use The wrong module is being used for a run where an external cooling device is attached Internal coolant system valve is stuck in the on or open position If an external cooling device is in use check the modules selected on the run sheet Use Chiller modules If no external cooling system is in use try to start the run as follows and place a service call Click OK in the error message box and try to start the run Open the Manual Control window and try to turn the pump on manually Error message Err Coolant Flow Failure Pump turned on and off three times to see if coolant flow was detected Open
421. ument status Data windows are Scan Gel Instrument windows are Status Electrophoresis History In addition to these windows a chronological comprehensive record of all significant system events including error and status messages is kept in a Log file All of these windows can be open at the same time however only one window can be active at any given time The Status Window During arun the current status of the instrument is viewed in the Status window The information in this window is updated approximately every three seconds Open the Status window by choosing Status from the Window menu Click arrows to Time remaining for the expand or collapse module being executed the window Status Laser Power mE Running Door mE Closed Total 03 30 00 Instrument State mmr Electrophoresis Power me Running Time Remaining 03 14 22 Electrophoresis Electrophoresis Electrophoresis Gel Laser Voltage kY Current m Power W Temperatur C Power mW 300 240 Green arrow is actual reading from instrument Gray box indicates the value set Total duration of the for the parameter by the module module being executed IMPORTANT The electrophoresis voltage gel temperature and laser power are setpoints Electrophoresis current and power are limits Data Collection Software 9 53 Artisa
422. upgrade only Stepped glass plates only 36 cm one set 4305384 for 96 lane upgrade only Clamps three 2 inch 4305386 use with 36 cm stepped glass plates P N 4305693 Glass Plate and Spacer Kit 401876 Includes two sets of 36 cm well to read glass plates and 36 cm spacers Rear Glass Plate 36 cm 401839 Front Glass Plate 36 cm 401840 Spacers two 36 cm 0 2 mm thick 401836 12 Glass Plate and Spacer Kit 401877 Includes two sets of 12 cm well to read glass plates and spacers Front Glass Plate 12 cm 401834 Rear Glass Plate 12 cm 401833 C 2 Parts and Accessories Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Combs and Overlays Wells Description Part Number 100 Sharktooth Comb 0 4 mm thick 1 8 mm center 4305385 96 lane 66 Squaretooth Comb 0 2 mm thick 66 well 402183 Plastic Overlay 66 well 402187 64 Sharktooth Comb 0 2 mm thick 64 well 402180 50 Squaretooth Comb 0 2 mm thick 50 well 402053 Plastic Overlay 50 well 402186 48 Sharktooth Comb 0 2 mm thick 48 well 402177 36 Sharktooth Comb 0 2 mm thick 36 well 401828 Squaretooth Comb 0 2 mm thick 36 well 401910 32 Sharktooth Comb 0 2 mm thick 32 well 401922 Squaretooth Comb 0 2 mm thick 32 well 401907 24 Squaretooth Comb 0 2 mm thick 24 well 401904 Sharktooth Comb 0 2 mm thick 24 well 401827 18 Sharktooth Comb 0 2 mm thick 18 well 402168
423. upper buffer chamber Upper Buffer Chamber Removing the Old Gasket ELECTRICAL SHOCK HAZARD The ABI Prism 377 contains a high voltage power supply Although the instrument has been designed with safety features in the door to disconnect the power supply when the door is open please follow procedures as prescribed As with any electrophoresis apparatus be careful during instrument operation and when handling electrodes and liquids Step Action 1 Disconnect the electrophoresis cable from the instrument and remove the buffer chamber 2 Place the buffer chamber on a piece of clean lab paper so the lens is not scratched and does not touch the work surface 3 Using the spatula remove the old gasket and sealant from the groove of the buffer chamber Be sure to remove all residual glue from the bottom of the groove IMPORTANT Do not use solvents to remove residual glue Solvents can damage the chamber GR1289 System Maintenance 8 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Installing the New Gasket To install the new gasket Step Action 1 Fit the new gasket into the groove to ensure a proper fit The rounded surface faces out The surface with the channel must contact the bottom of the groove in the chamber When properly fitted remove the gasket Trim the gasket if too long The gasket can be short within 1
424. uring amplification DNA Thermal Cycler 480 Push reaction tubes firmly into contact with block after first cycle Repeat amplification GeneAmp PCR System 9600 heated cover misaligned Align the heated cover so that white stripes align after twisting the top portion clockwise Poor thermal cycler performance Check instrument calibration 4 14 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Observation Possible Causes Recommended Actions Good signal from positive control but faint or no signal from sample DNA Sample contains PCR inhibitor for example heme compounds EDTA or certain dyes Quantitate DNA Dilute if possible in order to add minimum necessary volume Repeat amplification Wash the sample in an Amicon Centricon 100 column and repeat amplification Note For fragments smaller than 130 bp use the Amicon Centricon 30 column instead Add bovine serum albumin BSA to the PCR reaction mixture Use 8 16 ug BSA for every 50 uL PCR reaction volume Sample DNA is degraded Evaluate the quality and concentration of the DNA sample by Using the QuantiBlot Human DNA Quantitation Kit for human DNA Running an agarose yield gel If DNA is degraded or inaccurately quantitated reamplify with an increased amount of DNA Insufficient sample DNA added because of inaccurate quant
425. uring run warning displayed or Pump shuts down during run warning displayed and poor resolution due to gel running too cold Clog in coolant system Flush the coolant system to remove the clog and refill the water reservoir with deionized water and 5 0 antifreeze See Removing Coolant System Clogs on page 4 32 Rear panel LEDs are stuck in one pattern e g all on or all off Instrument memory cleared due to a power outage Corrupted firmware on instrument Perform a total reset or cold boot the instrument See Performing a Total Reset on page 4 22 Performing a Cold Boot on page 4 23 Computer cannot load a new firmware image onto the instrument or A new firmware image appears to have been downloaded however The instrument still does not operate The rear panel LEDs remain stuck in one pattern Instrument memory cleared due to a power outage Corrupted firmware on instrument Perform a total reset or cold boot the instrument See Performing a Total Reset on page 4 22 Performing a Cold Boot on page 4 23 4 8 Troubleshooting Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Observation Possible Causes Recommended Actions No current electrophoresis Incorrect TBE buffer formulation Use correct buffer concentrations 10X TBE in the gel
426. ving IMPORTANT Align the outside edge of each spacer with the outside edge of the plate Spacers must cover the notched areas of the plate Place water droplets along edges of plate to hold spacers in position Spacers Bottom of rear plate Pouring Gels 2 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com To mount glass plates in the gel cassette continued Step Action 6 Place the front plate on top of the rear plate and spacers so that the Gasket mark faces up hydrophobic area described on page 2 5 Bottom of the plates are flush Large notch in top of plate is oriented toward the top of the cassette see illustration in next step IMPORTANT To avoid difficulty pouring the gel always load the front and rear plates with the same side of the glass facing out Refer to Before Using New Glass Plates on page 2 5 for more information Keeping both plates together push the plates to the bottom of the cassette until the notches of the rear plates are seated firmly against the metal plate stops in the cassette Push the plates from the top to ensure firm contact r Bottom of plates must be flush Metal plate stops Run the tip of your finger along the bottom of the plates to make sure they are flush IMPORTANT The bottom edges of the plates must be flush with each other If the pla
427. w appliedbiosystems com www appliedbiosystems com AR Bes ys D Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 08 2001 Part Number 4307164B an Applera business Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com ABI PRism 377 DNA Sequencer Instrument Operation Quick Start Guide BSS BibEystems Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com rtisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Instrument Operation Chapter Contents In this Chapter The following topics are discussed in this chapter Topic See page Summary of Procedures for Performing a Run 3 2 Materials Required but not Supplied 3 2 Preparing the 1X Tris Borate EDTA TBE Buffer Solution 3 3 Preparing the Formamide Blue Dextran Loading Solution 3 3 Cleaning the Gel Plates Before Loading the Gel 3 4 Loading the Gel Into the Cassette 3 6 Installing the Gel Cassette and Lower Buffer Chamber 3 8 Performing a Plate Check 3 11 Skipping Lanes 3 14 Installing the Upper Buffer Chamber 3 15 Filling the Buffer Chambers 3 16 Instal
428. w them to air dry IMPORTANT To avoid damaging the electrode inside the buffer chambers rinse the buffer chambers only Do not scrub or clean them with a sponge 15 Follow the procedures listed in Chapter 2 Gel Preparation to Remove the gel from between the glass plates Clean the glass plates spacers and comb Instrument Operation 3 45 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Analyzing the Data For Sequencing Refer to the ABI PRisu DNA Sequencing Analysis Software User s Manual for Runs instructions on how to analyze data from a sequencing run For GeneScan Runs Refer to the ABI PRISM GeneScan Analysis Software User s Manual for instructions on how to analyze data from a GeneScan run Archiving and Printing Data from Runs Refer to Chapter 9 Data Collection Software for information on archiving and printing data 3 46 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com The Read Region What is the Read The read region is the area of glass scanned by the laser It is approximately 2 5 to Region 4 5 cm from the bottom of the glass and is 6 inches wide for standard and XL instruments 7 25 inches wide for 96 lane instruments 3inches wide for ABI PRISM 377 18 instruments Top of glass plates 4 5cm__ 2 5 cm C gt __ Re
429. within the System folder and click Open The Make Matrix dialog box should look like that shown below Note The numbers in the Start at and Points boxes are default values Your numbers may vary Make Matrix 1 dR6G matrix std Start at 19edTAMRA matrix std Start at 6 23edROHK matrix std Start at 21 dR110 matrix std Start at dRhod_BigDye Update File Instrument Comment Dye Primer Matrix Tag Terminator Matrix T Terminator Matrix a Click OK The computer makes the matrix When finished a dialog window appears with the message Make matrix successfully completed b Click OK Verify the instrument file by following the procedure Checking Instrument Files on page 7 16 Making Instrument Files for Sequencing 7 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Checking Instrument Files Introduction Instrument files can be verified two ways Using the DataUtility program Using analyzed matrix standard samples Using the This operation allows you to DataUtility Program 4 Check the quality of the matrices in the instrument file Verify that the matrix is appropriate for the chemistry being used Determine if the matrix is responsible for poor data To view the instrument file Step Action 1 Open the DataUtility program 2 From the Utilities menu choose Copy Matr
430. wn above sample would not be loaded in lanes 26 27 and 28 If the peaks are close to the edge of a lane we recommend skipping an extra lane Click Cancel and terminate the plate check If a sample sheet Then has not been completed proceed to Installing the Upper Buffer Chamber on page 3 15 has already been reopen the sample sheet and make the necessary completed changes to reflect the empty lanes If the sample sheet has already been imported to the run sheet a Open the sample sheet pop up menu and select lt none gt b Open the sample sheet pop up menu and reselect the sample sheet See Select the Sample Sheet on page 3 29 Proceed to Installing the Upper Buffer Chamber on page 3 15 3 14 Instrument Operation Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Installing the Upper Buffer Chamber Procedure To install the upper buffer chamber Step Action 1 Open the front panel of the instrument 2 Open the two top clamps holding the gel in the cassette 3 Rest the two tabs on the back of the chamber on the ears at the top of the front glass plate Ears of front glass plate Close the clamps to lock the chamber in place Connect the electrophoresis cable to the upper high voltage connection See Figure 3 1 on page 3 8 6 Proceed to Fill
431. x in the Data Utility Make Matrix dialog box The Measure Noise function of the program is used by Applied Biosystems Service personnel and is not discussed here Making Instrument Files for Sequencing 7 3 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Summary of Chemistries The Sequencing Five cycle sequencing chemistries are currently available to prepare DNA samples for Chemistries the ABI PRISM 377 DNA Sequencer Each chemistry requires the use of a specific virtual filter set as listed in the table below Chemistry Virtual Filter Set Fluorescein Rhodamine Dye Primer Rhodamine Dye Terminators A dRhodamine Terminators BigDye Terminators E BigDye Primers Color Guide for Data Display Windows The CCD Camera Data collection software collects the fluorescent signal from specific locations on a and Virtual CCD camera inside the instrument These locations correspond to different Filter Sets wavelengths of light The result is the same as using a physical filter to separate the light wavelengths This is referred to as a virtual filter since no physical filtering hardware is used Real Time Display On the real time displays the Scan and Gel windows the data collection program Colors Vary displays the light intensities color coded according to wavelength Blue green yellow and red in that order represent the wavelengths of the dy
432. xist in the raw data 2 Backup the raw sample files for the standards 3 Make a new instrument file using the DataUtility program Note You can also add matrix data to an existing instrument file See Adding or Replacing a Matrix in an Existing Instrument File on page 7 20 4 Verify the accuracy of the new instrument file by using it to analyze each matrix standard file the raw data See Making an Instrument File for Virtual Filter Set E from Matrix Standards on page 7 10 5 Store the new instrument file See Storing and Backing Up the Instrument File on page 7 19 An instrument file can contain a Dye Primer matrix a Taq Terminator matrix and or a T7 Sequences Terminator matrix The instrument file for virtual filter set A must contain a dye primer matrix even if this chemistry will not be used Otherwise the data collection software will not function properly This instrument file can also contain the matrix information for the Rhodamine Dye Terminators Chemistries used with virtual filter sets A and E are listed on page 7 4 Perform a run on the instrument using the matrix standard samples required to create the instrument file IMPORTANT Do not configure the run sheet to automatically analyze the matrix standard samples Select lt none gt for the Instrument File on the sample sheet If necessary deselect the Auto Analyze boxes on the run sheet by clicking the box to remove the X Proc
433. xternal power supply Determining Intake To identify the intake and outlet flow tubes on the instrument and Outlet Step Action 1 Turn on the instrument 2 Open the right panel of the instrument where the water bottle is mounted 3 Unscrew the water bottle and slowly pull it down to expose the ends of the tubes but do not remove the bottle Observe the direction of water flow from the tubes into and out of the bottle to identify the intake and outlet tubes Note Water coming into the bottle is from the instrument outlet and water drawn out of the bottle goes into the instrument intake Some instruments have a small cutout at the bottom of the water intake tube Turn off the instrument Disconnecting the To disconnect the internal pump Internal Pump Step Action 1 Remove the water bottle and pull down the intake and outlet tubes to remove them 2 Store the tubes and water bottle for later use 3 Step to the rear of the instrument and turn the external valve so that the arrow points down Figure 1 on page B 3 Cut two pieces of polyurethane tubing approximately three feet one meter long Insert one end of a piece of tubing into the intake port on the instrument Repeat step 5 with the second piece of tubing in the outlet port Attaching the To attach the external water bath to the instrument External Water Bath Step
434. y Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Folders Inside the Description of folder in the ABI PRISM 377 folder ABI PRISM 377 Folder 9 4 Data Collection Software Folder Name Description of Contents Runs Contains an individual run folder for each run sheet created At the end of a run the individual run folder will typically contain a run gel and log file generated by data collection software and sample files generated by the analysis software Sample Sheets Contains each sample sheet created Modules Contains the standard module files These files contain the commands used to operate the instrument for plate checks preruns and runs Chiller Modules Contains the module files used for plate checks preruns and runs for subambient temperature operation of the instrument Firmware Image Contains the firmware that resides on the instrument ABI PRISM 377 Collection The data collection software program file The ABI folder also referred to as the Settings folder is located in the System Folder on the Macintosh hard drive The ABI folder contains the following files File Type Description DyeSet Primer Also referred to as mobility files Contain the information used to compensate for differences shifts in sample mobility See DyeSet Primer Files on page 9 49 for more information Comb No longer us
435. y Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com primers dye BigDye 7 4 fluorescein rhodamine 7 4 printing automatic printing after analysis 9 58 files setting up for printing 9 58 sequence files 9 60 printing data from runs 3 46 project names how to define 3 25 purpose of 3 25 protein sequences analyzing with INHERIT software 1 21 protocols chemical abbreviates used A 9 factors that affect read lengths A 4 list of recipes and recommendations A 2 Long Ranger gels 10 ammonium persulfate A 15 4 75 Long Ranger Singel A 16 5 Long Ranger gel for SSCP A 17 not for SSCP A 16 to A 17 ingredients and run conditions A 15 MDE gel protocol A 18 PAGE PLUS gels ingredients and run conditions A 13 protocol A 14 Polyacrylamide gels gel solution protocols A 19 to A 21 ingredients and run conditions A 11 protocol A 11 to A 12 sequencing chemistries A 3 pull up peaks 6 3 pump off function description of pump on function description of purity effect of urea impurity A 6 3 49 3 49 Q quality temperature and gel quality A 8 QuantiBlot kit when to use 4 15 quitting Data Collection software 9 59 R read lengths factors that affect A 4 read region description of 1 15 read region description of 3 47 reagents purity how it affects gel quality A 5 to A 6 storing A 23 rebuilding the desktop 8 18 reproducibility affects of acrylamide impurity A 5 buffer impurity A 6 vacuum strengt
436. ysis Software User s Manual for more information Sample sheets can be printed Data Collection Software 9 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Preparing a Sequencing Sample Sheet Save Time by Setting Sample Sheet Preferences Entering Information Opening a New Sample Sheet IMPORTANT Do not mix sequence and GeneScan analysis samples on the same sample sheet or in the same run If the same type of run is performed repeatedly on the same instrument you can reduce the time spent setting up sample sheets by setting sequencing sample sheet default preferences Setting sample sheet preferences means setting the default value of certain fields to the values used most often The following fields on sequencing sample sheets can be set as preferences DyeSet Primer Instrument File Once these preferences have been set the preferred values appear automatically on each new sequence sample sheet Preferences can be changed as often as necessary either by setting new preference values or by opening the pop up menus and manually selecting new values Instructions for setting sequencing sample sheet default preferences are located in Chapter 5 Setting Preferences See How to Enter Information on Sample Sheets on page 9 26 Procedures include Applying the same parameter to all fields in a column Copying information from one field or row to anothe
437. ystem file This file Is invisible to users Keeps track of where everything is located on the hard disk Grows larger over time if it is not rebuilt If the Desktop system file grows too large the computer s response time will slow to unacceptable levels Therefore we recommend rebuilding the desktop Once a month After installing new software If you get a system error message with a bomb after inserting a disk Procedure To rebuild the desktop Step Action 1 Hold down the Command and Option keys and choose Restart from the Special menu 2 Continue to hold down the two keys until the message Are you sure you want to rebuild the desktop file appears on the screen 3 Click OK Deleting Data Files Overview We recommend removing unnecessary files from the hard disk before starting the 8 18 System Maintenance data collection program For example the ABI PRISM 377 data collection and analysis software programs create large data files gel files sample files sample sheets and run sheets that accumulate on the hard disk Data files include gel files sample files sample sheets and run sheets These files can quickly fill all the available memory on the computer Backup data files and other files that are not used regularly and then delete the original files from the hard disk to reclaim memory for future work This practice will ensure that enough disk space is available for new data files Artisan Technology G
438. ze Standard and select the appropriate file Open the pop up menu for Analysis parameters and select the appropriate file Note Refer to the ABI PRISM GeneScan Analysis Software User s Manual for more information on matrix files analysis parameters and size standard files To specify that data be printed automatically at the end of each run select the check box labeled Auto Print If Then you are finished click OK to save your changes or Cancel to quit without making any changes you wish to change more preferences open the Page pop up menu and select another Preference Setting Preferences Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com General Settings About General The General Settings window allows you to specify the following Setting Preferences Setting Description Instrument Name Used to track which instrument a gel is run on The name is recorded in the Gel and Log files for each run Global Serial Number Increments with each run The number can be automatically added as a suffix to all Sample File names This feature is set via the File Names preference window sample file name Suppress Left Right Averaging Do not use this feature Current versions of the ABI PRISM 377 DNA sequencing and GeneScan analysis software cannot process unaveraged data This feature may be added to future ve
439. zing the data with baselining deselected in the Analysis Parameters dialog box As shown in Figure 6 4 low data points are apparent as troughs in one color beneath peaks in another lt Two large black peaks Elevated green baseline Figure 6 3 Characteristic appearance of an elevated baseline caused by a bad or incorrect matrix 6 4 Making Matrix Files for GeneScan Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Bavaro Figure 6 4 The data of Figure 6 3 before baselining What To Do If You If problems related to bleedthrough peaks or to an elevated interpeak baseline appear Have Matrix with any regularity you should remake the matrix Apply the new matrix to the old Problems sample data and reanalyze the data Instructions for creating matrix files are listed under Creating a Matrix File for GeneScan Applications on page 6 6 Making Matrix Files for GeneScan 6 5 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Creating a Matrix File for GeneScan Applications Overview The matrix file contains the information necessary for software to correct the spectral overlap of the dyes in the virtual filter sets Once a matrix file has been created it can be used for subsequent runs performed With the same kit or chemistry On

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