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1. To change a module for a sample click the module you want to change Click the arrow that appears at the right side of the field to open the drop down list and select a different module The default setting for the injection list assumes that each sample will be injected once If you prefer multiple injections modify the Tube amp Sample Name column so that the sample appears multiple times To make multiple injections from one sample tube Step Action 1 In this example the original Sample Sheet will be modified to inject the sample in A3 twice 4 GeneScan Injection Sheet E x Sample Sheet Sample Sheet GSscreenshots v D D Run Pause i cancel Length to Detector fo cm Operator Inj Tube amp Sample Name Module Inj Secs ni v Run kv Run C Run Time Matrix File 24 A1 Sample 1 GS STR POP4 1 mL F md4 1l 5 15 0 15 0 60 Bogus Matrix Aaj 2 A3 Sample 2 GS STR POP4 1 mL F md4 5 150 150 24 Bogus Matrix 3 A5 Sample 3 GS STR POP4 1 mL F md4 5 150 150 24 Bogus Matrix 4 Bogus Matrix l 4 gt Injection Lists 5 5 To make multiple injections from one sample tube continued Step Action 2 For the injection you are modifying click the sample name in the Tube amp Sample Name column then click the arrow that appears next to th
2. Topic See Page Section Using the Data Collection Software 4 3 About the Data Collection Software 4 4 Manual Control 4 6 Raw Data Display 4 8 Run Status and History 4 11 Modules 4 14 Section Installation and Configuration of the Data Collection Software 4 17 Installing the Data Collection Software 4 18 Data Collection Software Preference Files 4 19 General Software Procedures 4 29 Using the 310 Data Collection Software 4 1 4 2 Using the 310 Data Collection Software Section Using the Data Collection Software In This Section This section covers the following topics Topic See Page About the Data Collection Software 4 4 Manual Control 4 6 Raw Data Display 4 8 Run Status and History 4 11 Modules 4 14 Using the 310 Data Collection Software 4 3 About the Data Collection Software Overview The ABI PRISM9 310 Data Collection Software is installed by the Field Service Engineer on the computer when setting up the ABI PRISM9 310 Genetic Analyzer in your laboratory If you need to reinstall the Data Collection software refer to Installation and Configuration of the Data Collection Software on page 4 17 The Data Collection software provides instructions to firmware running on the instrument and displays instrument status and raw data in real time As the instrument records sample fluorescence on the detection system hardware the Data Collection software running on th
3. 2 Align the steel shafts with the two large holes of the block 3 Before sliding the gel block all the way to the back of the shafts align the anode buffer reservoir valve with the activator arm Slide the U shaped end of the activator arm into the collar at the top of the valve s plunger Lift the plunger about 3 mm if necessary and slide the gel block until flush against the instrument Filling the Gel Block To fill the gel block channels with polymer Step Action 1 From the Function menu of the Manual Control window select Buffer Valve Close This closes the pin valve at the anode buffer reservoir on the gel block Open the waste valve below the syringe manually Press the syringe plunger until a drop of polymer forms on the bottom of the waste valve This removes the air bubbles at this valve site and uses about 0 1 mL of polymer Close the waste valve manually From the Function menu of the Manual Control window select Buffer Valve Open This opens the pin valve at the anode buffer reservoir on the gel block Press the syringe plunger until polymer fills the polymer channel in the block This removes all of the air bubbles from the polymer channels and uses about 0 1 mL of polymer IMPORTANT There should be no air bubbles in the gel block channels From the Function menu select Buffer Valve Close Move the syringe drive toggle to the right to p
4. 2 7 81 8 49 26 Vial A1 inject 5 secs 15 8kU run 28 mins at 15 8kU 66 C 2 7 81 9 66 68 EP 14 9kU 8 9mf 68 laser 9 9mW syringe 463 lt 2 7 01 9 26 26 EP 14 9kU 6 6mA 66C laser 9 9mWV syringe 463 2 7 981 9 26 26 Points collected 5468 2 7 81 9 26 26 Injection 1 End 2 7 81 9 26 26 Injection 2 hex green 2 7 81 9 26 26 Module GS STR POP4 1 mL D md 2 7 81 9 26 26 Vial A2 inject 5 secs 15 8kU run 28 mins at 15 8kU 66 C 9 32 52 EP 14 9kU 6 9mA 68C laser 9 9mWV syringe 466 9 52 59 EP 14 9kU 6 6mA 68C laser 9 9mWV syringe 466 9 52 59 Points collected 5468 9 52 59 Injection 2 End 9 53 08 Injection 3 hex yellow 9 53 88 Module GS STR POP 1 mL D md 9 53 08 Vial A3 inject 5 secs 15 8kU run 28 mins at 15 6kU 66 C 9 59 15 EP 14 9kU 8 9mf 66C laser 9 9mW syringe 469 18 19 31 EP 14 9kU 6 6mA 66C laser 9 9mW syringe 469 16 19 31 Points collected 5468 18 19 31 Injection 3 End 18 19 32 Injection 4 rox red 18 19 32 Module GS STR POP4 1 mL D md4 18 19 32 Vial A4 inject 5 secs 15 8kU run 28 mins at 15 8kU 66 C 18 25 56 EP 14 9kU 6 9mA 66C laser 9 9mW syringe 413 18 56 85 EP 14 9kU 6 6mA 66C laser 9 9mWV syringe 413 The information in the file is formatted as follows XXX month day year hour minute second Description of event The entry in the first column xxx is variable The possible entries are Entry Meaning System start system stop or file created gt Mes
5. The recommended quantities for sequencing reactions are shown in the table on page 6 36 This section provides information regarding PCR template preparation but it is not meant to be a detailed guide to PCR amplification General information on PCR amplification can be found in the Guide to PCR Enzymes and in the product inserts included with GeneAmp PCR reagents For PCR amplification use GeneAmp PCR Instrument Systems and GeneAmp PCR Core Reagents Cycle sequencing has been found to provide the most reproducible results when sequencing PCR templates Although PCR fragments can be difficult to denature with traditional sequencing methods cycle sequencing provides several chances to denature and extend the template which ensures adequate signal in the sequencing reaction 6 34 Setting Up DNA Sequencing Experiments About DNA Template Quality Using Control DNA Sequencing Standards Poor Template Preparation Contamination Always use a control DNA template when preparing your sequencing reactions This can help determine whether failed reactions are the result of poor template quality or sequencing reaction failure We recommend M13mp18 as a single stranded control and pGEM 3Zf as a double stranded control All Applied Biosystems DNA sequencing kits provide pGEM control DNA and a 21 M13 primer A sample standard verifies operation of the instrument s hardware just as the control template verifies the correct prepa
6. 1 Hold the Tray and Gel buttons down for 10 seconds while you turn on the 310 Genetic Analyzer This deletes the firmware and clears the RAM The three lit status lights indicate the absence of firmware Restart the computer and open the Data Collection software A dialog box will appear as the firmware reloads Open the Manual Control window and enter the calibration values again manually for CCD Pixel Position X horizontal CCD Pixel Position Y vertical Syringe Pump Force Syringe Max Travel or select Calibration File Send Select Syringe Home and click Execute In the Manual Control window select Autosampler Home X Y and click Execute Autosampler Home Z and click Execute Calibrate the autosampler See Calibrating the Autosampler on page 3 4 Wait 10 minutes after a clear memory reset before starting a run This allows the temperature circuitry to calibrate 3 48 Operating the Instrument Hardware About Recovering The 310 Genetic Analyzer will not recover from a power failure from a Power To reduce the chance of power failure it is recommended that you install a UPS unit Failure for the computer Contact technical support for recommendations for UPS units IMPORTANT Under power down or power fail situations the autosampler drops exposing an open end of the capillary to air If power was lost for more than 30 minutes check the EPT file during the n
7. D lt 9 o Note For a four dye set the window displays a 4x4 matrix of numerical values For a five dye set the window displays a 5x5 matrix of numerical values 2 Confirm the following The numbers on the diagonal Blue against Blue Green against Green etc must all be 1 00 The numbers off the diagonal are less than 1 00 Setting Up Fragment Analysis Experiments 7 27 Inspecting the To check matrix quality Matrix Quality Step Action 1 From the Project containing your matrix standard sample files open the Analysis Control window amp Install Run Filter Set F_prj Analysis Control Analyze Print Results Sample File Size Standard ia Parameters ia 1 A1 Matrix Standard 1 Size Standard al sAnalysis Parameters 2 43 Matrix Standard 2 Size Standard al Analysis Parameters 3 45 Matrix Standard 3 Size Standard al Analysis Parameters 9 A7 Matrix Standard 4 Size Standard at lt Analysis Parameters ol lt Analysis Parameters Z In the Analysis Control window select the samples containing the matrix standards Select the matrix standard sample files From in the Project menu select Assign New Matrix then select the matrix file Apply the new matrix file to the sample file used to generate it 5 From the Settings menu use the Set Analysis Parameters dialog box to set the Analysis Range
8. Label one of the buffer vials as Buffer and fill it to the line with 1X Genetic Analyzer buffer with EDTA Cap the vial insert the septum and place it in position 1 on the autosampler This serves as the cathode buffer Label the other glass buffer vial as H O and fill it to the line with filter sterilized deionized water Cap the vial insert the septum and place it in position 2 on the autosampler If the 1 5 mL Eppendorf tube has a lid attached cut the lid off Fill the tube completely with filter sterilized deionized water and place it in position 3 on the autosampler Note Do not use a screw cap tube The screw cap tubes are too high to clear the electrode and capillary Attach the anode buffer reservoir containing the buffer solution to the gel block 3 8 Operating the Instrument Hardware Loading the Samples The following steps describe how to load the samples on the autosampler To load samples in the autosampler tray 1 Press the Tray button to bring the autosampler platform forward Put the tray on the platform Press the Tray button to move the autosampler back Note For information about inserting the 96 well tray see Inserting the 96 Well Tray on page 3 34 Operating the Instrument Hardware 3 9 Buffers and Polymer When to Replace Use the following table Reagents Reagent Replace Cathode and Anode buffers Every 48 ho
9. Step Action 8 Click Start 9 After the module has run again note the position of the plunger and subtract it from the initial value The module should consume 4 7 uL of polymer per 10 minute fill Check for leaks if more than 7 pL of polymer was consumed Correcting Leaks at the Syringe To correct leaks at the syringe Step Action 1 Replace the syringe if polymer leakage is Excessive past the plunger s seal Between the metal collar and the glass barrel Loss of only a few microliters of polymer out of a full syringe is normal and acceptable If polymer has leaked from the base of the glass syringe check for the presence of two O rings and a plastic ferrule O rings Metal collar and glass barrel ct rmm 0 05 0 1 0 15 0 2 0 25 Replace the O rings if they are worn Correcting Leaks at the Valve to the Buffer Reservoir To correct leaks at the valve to the buffer reservoir Step Action 1 Remove the gel block and rinse it with warm pure water 2 Flush the gel block channel to the anode buffer reservoir with warm pure water Buffer reservoir valve B Ho ME Cel LO O TI GR1058 Channel to the anode buffer reservoir Attach a 5 mL plastic syringe filled with warm pure water to the gel block 3 12 Opera
10. Things You Can Do During a Run Using the Injection List to Add Samples to a Sample Sheet Mid Run To add samples to an active sample sheet during a run Step Action 1 From the injection list click the page icon C next to the Sample Sheet drop down list This opens the currently running sample sheet Note You cannot change or delete the samples in the sample sheet which are used in the injection list These samples will appeared grayed out when you reopen a sample sheet to make additions S D3AppliedBio 310 Sample Sheets Sample Sheet 042301 gss _ Sample Hame Collection Name A1 one none snone gt none none j none none Add a new sample by typing a name in the Sample Name field and filling in the rest of the information for the row When finished select Save from the File menu then close the sample sheet In the Injection List window your new samples are now available for addition into the injection list Injection Lists 5 9 Adding New To add new samples to the injection list during a run Samples to an Step Action Injection List Mid Run 1 You can add new injections anywhere in the injection list following the currently running injection a Select an injection b Select Insert from the Edit menu A new line will be
11. 6 FAM 5 FAM Green dR6G vic HEX vic TET JOE Yellow dTAMRA NED NED NED HEX NED Red dROX PET ROX ROX TAMRA ROX Orange LIz LIZ 4 10 Using the 310 Data Collection Software Run Status and History Monitoring Run You can monitor the status of your run using the Data Collection software The Data Status and History Instrument Run Status Log File Collection software collects raw data and allows real time run monitoring in four software windows that show Instrument run status Sample raw data Instrument log file Electrophoresis history During a run the current status of the instrument is displayed in the Status window To view the status of your run select Status from the Window menu Estatus EX S Instrument State EEE idle Laser ME Running Electrophoresis Power BI Off Door W Closed Y injection Function idle 48 Tube Autosampler 10 Time Remaining 00 00 00 Buffer Valve Closed GelPump At Set Total Time 00 00 00 v Electrophoresis Electrophoresis Gel Laser Voltage kV Current pA Temperature C Power mW 15 99 39 8 6 4 2 0 The Status window displays Current state of the instrument Time remaining in the current function Actual electrophoresis voltage heat plate temperature and laser power Set points for the electrophoresis voltage heat plate temperature and laser power 9 9
12. C above room Temperature temperature Note There is no active cooling system on the 310 Genetic Analyzer You cannot set the heat plate to a temperature lower than the ambient temperature About You can change the running time for special requirements For example you can Electrophoresis shorten the data collection time if you only need information about short extension Running Time Products 6 18 Setting Up DNA Sequencing Experiments Selecting the Module and Dye Set Primer File Overview Updated Files Modules Mobility Set Primer Files Reference Table for Dye Set Primer Files and Modules DNA sequencing on the 310 Genetic Analyzer requires you to have these types of files Module Basecaller Dye set primer Matrix You select the particular files appropriate to your experiment on the sample sheet and injection list The module basecaller and dye set primer files are supplied with the Data Collection software Updated files can be obtained from Applied Biosystems Web site http www appliedbiosystems com Applied Biosystems Technical Support or from your local field applications specialist call your local sales office for more information For new applications a disk containing the files is often included with the reagent kit Matrix files are all created using the Sequencing Analysis software See the ABI PRISM DNA Sequencing Analysis Software User Guide Modules are selected in the Modu
13. Run temperature 9 9 9 Run time You can edit these parameters by selecting a field and typing in a new value This changes the run conditions for one run It does not permanently alter the module For information about editing a module see Editing Modules on page 4 14 Change Sample Sheet Items from the Injection List The following files on a sample sheet can be changed after viewing the sample sheet from the injection list Dye set primer Matrix The electrokinetic injection time and voltage can be varied to regulate the amount of DNA brought into the capillary for analysis The objective is to inject just enough signal into the capillary for adequate peak height good signal to noise while minimizing the peak widths There are no specific requirements for the voltage and current during the electrokinetic injection Injections at 200 volts cm are not distinguishable from injections at 25 volts cm however a lower voltage and current are preferable since the timing of the injection is more accurate Typically a voltage of 50 volts cm is used for the electrokinetic injection At this value injection times of 5 to 60 seconds are required to obtain adequate peak height depending on the application Note Excessive injection time can cause loss of resolution Setting Up DNA Sequencing Experiments 6 17 Electrophoresis Resolution and basecalling are better at lower field strengths Increasing the field Running Vo
14. cee he 4 11 LOg Blei Seas awe et wot Ses CERES TR BEL ERS Ah Deine Deer een es 4 11 Electrophoresis History iil kde Re ade er Rx REESE ERE 4 13 Adjusting the Voltage Current Laser and Temperature Scales 4 13 Modules omi ee REI CULA TENSES LA e ELS RAE ee ERROR A 4 14 Editing Modules 0 cee cee eee hem rem hers 4 14 Editable Parameters oes eR bch te RR Ne tEERERRE S EUER SENE 4 14 About Module Parameters isses cere ee Rm ry RE RR ORCI 4 15 Section Installation and Configuration of the Data Collection Software 4 17 mhis Section incus tenise eri ieni HAG Rie ER ST e RENDERE ERCEPES CSS 4 17 Installing the Data Collection Software sss 4 18 The Data Collection Software Installer Disk sleleeseeeeeeeesns 4 18 Reinstalling the Data Collection Software lsleeeeeee eese 4 18 Installing Modules and Dye Set Primer Files 0 000 00 0000 4 18 Data Collection Software Preference Files 2 0 cece eee eee eee eee 4 19 OVeEVIGW s i eR CROU eU REA N AER See GAM Ace aOR A Sea BO Re 4 19 About the Preferences Window 0 cece eee eect tenes 4 19 Folder Locations Preferences ys sirae nann e cece cette ee 4 20 Pile Names Preferences orte e CERRO LES e e VR 4 21 Sequencing Sample Sheet Defaults Preferences 000000000 4 22 GeneScan Sample Sheet Defaults Preferences 0 00000 e eee eee 4 23 Sequencing Injection List Defa
15. concentrations can lower the Extra peak of size frequency of nontemplate n 1 nucleotide addition and vice versa Increasing the extension time at 72 C will increase the frequency of nontemplate nucleotide addition For more suggestions refer to the GeneScan Chemistry Reference for the ABI PRISM 310 Genetic Analyzer Troubleshooting 8 23 Limited Warranty To What This Warranty Applies To What This Warranty Does Not Apply Applied Biosystems Obligation 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 310 DNA Sequencer purchased by the Customer the Instrument will be free from defects in material and workmanship and will perform in accordance with the performance specifications contained in the Instrument User s manual that accompanies the instrument the Specifications During the Warranty Period if the Instrument fails to perform in accordance with such specifications Applied Biosystems will repair or replace the Instrument at no charge to the Customer subject to the conditions below This Warranty does not apply to the Instrument s valves reagent lines or performance unless the Customer uses only reagents and solvents supplied by Applied Biosystems or expressly recommended by Applied Biosystems or t
16. none none none none snone gt none none enone gt none none enone gt none none enone gt none none snone none none Jenone gt none none enone gt none none enone gt none none snone gt none none none none none none Note Drag the column markers at the top of columns to change their width Select 4 Dyes or 5 Dyes from the drop down list in the upper right corner based on the sample type In the Sample Name column enter the name of each of the samples Note The number in the first column corresponds to the location of the sample in the autosampler tray Note If there is no sample name entered the sample will not appear in the injection list In the Dye Set Primer column select the appropriate file for each of the samples from the drop down list See Selecting the Module and Dye Set Primer File on page 6 19 if you need help selecting the correct file To select a file a Click the field in the Dye Set Primer column none in the example screen above An arrow will appear at the right side of the field b Click the arrow to open the drop down list c Select the appropriate file from the list Note _ If there is no dye set primer file selected the sample will not be autoanalyzed In the Matrix File column select the matrix file for eac
17. 100 bp to 900 bp 401098 GeneScan 2500 ROX 100 bp to 2500 bp 401100 GeneScan 2500 TAMRA 100 bp to 2500 bp 401545 Consumables B 5 Fragment Analysis Use these part numbers to order fragment analysis consumables Consumables Part Numbers B 6 Consumables Part Consumable Purpose Number POP 4 Performance Optimized Polymer for electrophoresing DNA under 402838 Polymer denaturing conditions GeneScan Polymer Polymer for electrophoresing DNA under 401885 non denaturing conditions 10X Genetic Analyzer Buffer Running buffer for electrophoresis 402824 with EDTA for use with POP 4 Index Numerics 1X reactions 6 39 310 Genetic Analyzer back of instrument 2 5 On Off switch 2 6 reset button 2 6 serial connections 2 6 behind the doors 2 7 to 2 11 autosampler region 2 11 detection region 2 10 gel block region 2 8 to 2 9 cleaning general recommendations 3 42 front of instrument 2 4 doors 2 4 operating principles 2 19 to 2 22 preferences 4 19 to 4 27 Dye Indicators preferences 4 27 File Location preferences 4 20 File Names preferences 4 21 General Settings preferences 4 26 Injection List preferences 4 24 to 4 26 Sample Sheets preferences 4 22 to 4 23 preparing forarun 3 2 recovering from power failure 3 49 resetting 3 46 to 3 48 clear memory reset performing 3 48 clear memory reset recording calibration data 3 48 cold boot performing 3 47 firmware about 3 46 soft reset performi
18. By telephone or fax Through the Applied Biosystems web site 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 Technical Documents following the telephone information below To Contact Technical To contact Applied Biosystems Technical Support by e mail for help in the following Support by E Mail product areas Product 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 corelab appliedbiosystems com Synthesis Biochromatography tsupport appliedbiosystems com PerSeptive DNA PNA and Peptide Synthesis systems FMAT 8100 HTS System CytoFluor 4000 Fluorescence Plate Reader Mariner Mass Spectrometers Voyager Mass Spectrometers MassGenotyping Solution 1 MGS1 System LC MS support sciex com Applied Biosystems MDS Sciex Chemiluminescence Tropix tropix appliedbiosystems com To Contact Technical To contact Applied Biosystems Technical Support in North America use the telephone Support by Or fax numbers in the table below Telephone or Fax North America Note To schedule a service call for other support needs or in case of an emergency
19. Collection Info Collection information is used by the Sequence Collector software The collection Preferences names entered in the Collection Info tab will appear in the Collection Name drop down list in the sample sheet Only the sample files with collection names can be uploaded into the Sequence Collector software Preferences Delete mov x oa Set the following Collection Info preferences Preference Description Add Row To add a new entry click the Add Row button and fill in the collection information Delete Row To delete an entry select the entry you want to delete and click the Delete Row button 4 28 Using the 310 Data Collection Software General Software Procedures How to Find Files When files are stored on a computer you can organize them by saving each one ina folder with related files The organization can be taken a step farther by gathering related folders together inside yet another folder If Then you want the location of a Double click the My Computer icon on the computer desktop a file that is not open b Double click the drive and folder icons to search the computer a file is open the name of the file and its location are displayed at the top of the window The drive designation is displayed first Folders and sub folders are listed separated by backslashes Y Keyboard Shortcuts If there is a keyboard shortcut for a comma
20. Procedure See Page Turning On the Genetic Analyzer Preparing the Polymer and the Electrophoresis Buffer Cleaning the Gel Block Filling the Gel Block Cleaning the Electrode Installing the Capillary Calibrating the Autosampler Testing the Capillary Window Filling the Buffer Reservoir Cleaning the Syringe Filling the Syringe Installing the Syringe Homing the Syringe Preheating the Heat Plate What to Do Next i you are running a Then DNA sequencing experiment Proceed to Chapter 6 Setting Up DNA Sequencing Experiments to prepare the sample sample sheet and injection list Fragment analysis experiment Proceed to Chapter 7 Setting Up Fragment Analysis Experiments to prepare the sample sample sheet and injection list 3 2 Operating the Instrument Hardware Section Procedures for Setting Up and Maintaining the Instrument In This Section This section covers the following topics Topic See Page Autosampler 3 4 Buffers and Polymer 3 10 Capillary 3 14 Electrode 3 23 Gel Block 3 27 Heat Plate 3 30 Sample Trays 3 31 Syringes 3 35 Operating the Instrument Hardware 3 3 Autosampler Cleaning the When to Clean the Autosampler Autosampler Glean the autosampler once a week How to Clean the Autosampler To clean the autosampler remove dried buffer from the autosampler with a water
21. The analysis parameters file holds the default start and stop point for data analysis the default peak height and width threshold and the default size calling method Size standards The size standard file holds the results of a run performed with fragments of known length This file can be used to analyze other samples run with the same size standard under the same conditions to determine the size of fragments of unknown length Where the Data is The Data Collection software creates a Run folder automatically to hold the sample Stored files when you start a run The data from each sample is stored in a sample file Sample files from the same run are stored in the same Run folder Using the 310 Data Collection Software 4 5 Manual Control Overview Manual Control allows you to manually operate Functions Test modules Functions are specific mechanical tasks such as moving the autosampler moving the syringe and turning the laser on and off Manual Control also allows you to edit modules See Editing Modules on page 4 14 Manual Control From the Manual Control window you can Functions Present and return the autosampler tray Home the X Y and Z axes of the autosampler Position the autosampler Open and close the buffer valve Turn electrophoresis on and off Set electrophoresis voltage Turn the laser on and off Run the laser Set laser power Put laser on standby Home the syringe Move the syri
22. To move the capillary before turning on the instrument Step Action 1 Position the capillary end 0 5 mm below the end of the electrode 2 Securely tape the capillary to the heat plate in this position 3 Turn on the power to the instrument Storing the Capillary Off the Instrument To store the capillary off the instrument Step Action 1 Label the capillary with the number of runs performed A capillary can be used for up to 100 runs Keep track of the number of runs performed on each capillary Maintain the capillary ends in two sample tubes filled with sterile deionized water and closed with septa Water evaporates very slowly from the tubes Check them every week and add water if necessary Always clean the gel block after removing the capillary 3 20 Operating the Instrument Hardware Removing the Capillary for Short term Storage Use this procedure if the capillary will be reused within 1 week To remove the capillary for short term storage Step Action 1 Open the Data Collection software if not open already 2 From the Manual Control window run the Seq Fill Capillary module to remove any sample still in the capillary 3 Leave the polymer in the capillary 4 Remove the capillary by following the installation steps in reverse order Clean the gel block Removing the Capillary for Long term Storage To remove th
23. dial 1 800 831 6844 then press 1 Product Product Area ABI Prism 3700 DNA Analyzer Telephone Fax 1 800 831 6844 1 650 638 5981 then press 82 1 800 831 6844 press 2 then press 1a 1 800 831 6844 press 2 then press 24 DNA Synthesis 1 650 638 5981 Fluorescent DNA Sequencing 1 650 638 5981 About This Guide 1 9 1 10 About This Guide Product Product Area Fluorescent Fragment Analysis including GeneScan applications Telephone 1 800 831 6844 press 2 then press 34 Fax 1 650 638 5981 Integrated Thermal Cyclers ABI PRISM9 877 and Catalyst 800 instruments 1 800 831 6844 press 2 then press 4a 1 650 638 5981 ABI PRISM 3100 Genetic Analyzer Peptide Synthesis 433 and 43x Systems 1 800 831 6844 press 2 then press 64 1 800 831 6844 press 3 then press 14 1 650 638 5981 1 650 638 5981 Protein Sequencing Procise9 Protein Sequencing Systems 1 800 831 6844 press 3 then press 24 1 650 638 5981 PCR and Sequence Detection 1 800 762 4001 then press 1 for PCRa 2 for TagMan applications and Sequence Detection Systems including ABI Prism 7700 7900 and 57008 6 for the 6700 Automated Sample Prep Systema or 1 800 831 6844 then press 5a 1 240 453 4613 Voyager MALDI TOF Biospectrometry Workstations Mariner ESI TOF Mass Spectrometry Workstations MassGenotyping Solution 1 MGS1 System Bioc
24. the other colors are not flat under the peaks the matrix is poor 8 If the Then matrix is good Save the matrix file for future use matrix is poor Reanalyze the matrix run If this does not improve the matrix data run new matrix standards 6 32 Setting Up DNA Sequencing Experiments Section Useful Information for Preparing DNA Sequencing Samples In This Section This section covers the following topics Topic See Page About DNA Template Preparation 6 34 About DNA Template Quality 6 35 About Primer Design 6 37 About Preparing Sequencing Reactions 6 38 About Cycle Sequencing 6 40 About Preparing Extension Products for Electrophoresis 6 41 Setting Up DNA Sequencing Experiments 6 33 About DNA Template Preparation Reference Document DNA Template Preparation PCR Templates Refer to the following documents for more information about the material covered in this section Automated DNA Sequencing Chemistry Guide for more information on DNA template preparation and specific protocols Comparative PCR Sequencing a Guide to Sequencing Based Mutation Detection for more detailed information about PCR sequencing The DNA purification method you use can affect the quality of the template preparation Some recommendations for purifying DNA templates are given below Prepare adequate template to check purity quantitate the DNA accurately and perform the sequencing reactions
25. 0 eee cee eee 5 7 Changing Run Parameters 02 tes ec eects 5 7 Changing the Order of the Samples ina Run 0 cece eee eee 5 7 Things You Can Do During a Run 1 cee 5 9 Using the Injection List to Add Samples to a Sample Sheet Mid Run 5 9 Adding New Samples to an Injection List Mid Run 0 0 5 10 Adding Samples to the Instrument Mid Run llle esee 5 11 Setting Up DNA Sequencing Experiments Introduction ics e nee prede A gala abate ORDER M Eg REED REIR 6 1 In T his Ch pter i eo eene Rane a eres eter e ee 6 1 About this Chaptetz iicoesre hls pb DER RE RP VREPERSMRTEPUDEM 6 2 Section Setting Up a DNA Sequencing Run eee 6 3 In This Section corte eLERRREPRESR URSI MR S ERE EPIS ROREM 6 3 About Sequencing Analysis lleseeeeeeeeee e eens 6 4 PCR and Cycle Sequencing 00 cee ee 6 4 Cycle Sequencing Using Terminators 0 0 0 eee eee ee 6 5 DNA Sequencing Chemistries 0 0 0 0 cece cee eee eee 6 5 Description of Chemistry Table Levels ee eee 6 5 310 Chemistry Options Table sslsseeeeeeee A 6 6 Performing a DNA Sequencing Experiment 0 00 0 cece eee eee 6 7 Materials Required oos a ete e eer ee EO PUE P ERREUR ee 6 7 Software Required vius eee TIONES E EC SPENT Rd en 6 8 Summary of Procedures ovana n e A E E n 6 8 Denaturing and Loading Samples 0 0 eee cece ee 6 9 Abo
26. 02 Mother GS STR POP4 1 mL 15 0 15 0 24 Bogus Matrix mtx 1347 10 Son CS STR POP4 1 mL i 15 0 24 Bogus Matrix mtx Matrix Standard 1 les STR POP4 1 mL 15 0 24 Bogus Matrix mtx Matrix Standard 2 les STR POP4 1 mL 15 0 24 Bogus Matrix mtx Matrix Standard 3 GS STR POP4 1 mL 15 0 24 Bogus Matrix mtx Matrix Standard 4 GS STR POP4 1 mL 15 0 24 Bogus Matrix mtx The following is an example of an injection list without matrix standard samples EdceneScan Injection Sheet Untitled 1 xl Sample Sheet Sample Sheet gss ADO P Run Pause M concel Length to Detector 30 cm Operator nj Tube amp Sample Hame Module Inj Secs Inj kV Run kv Run C Run Time Matrix File 5 E 60 T 1347 15 Maternal Gr les STR POP4 1 mL 15 0 24 Bogus Matrix mtx aj 1347 02 Mother IGS STR POP4 1 mL 150 60 24 Bogus Matrix mtx 2 3 1347 01 Father CS STR POP4 1 mL 150 f 60 24 Bogus Matrix mtx 4 1347 10 Son GS STR POP4 1 mL 150 f 60 24 Bogus Matrix mix v JE E Note Drag the column markers at the top of columns to change their width Complete the injection list a Enter your name as the Operator b Open the Module drop down list for each sample and select a module Note When a 5 dye sample sheet is imported into an injection list only 5 dye modules are available in the drop down
27. 3 Tape the capillary to the heat plate with thermal tape to secure the position of the capillary labelling mark colored capillary mark relative to the detector plate For a diagram showing how the capillary is taped to the heat plate see Detection Region on page 2 10 4 Close the laser detector door to secure the position of the capillary window Operating the Instrument Hardware 3 17 Positioning the Capillary Near the Electrode The capillary and cathode electrode should be as close to each other as possible The capillary should protrude beyond the electrode no more than 0 5 mm To position the electrode end of the capillary Step Action 1 Thread the capillary through the capillary hole in the electrode thumbscrew until it protrudes past the tip of the electrode by about 0 5 mm maximum Tape the capillary to the heat plate with thermal tape to secure the position of the capillary tip relative to the electrode Tape the capillary just above the electrode thumbscrew and just above the detector door IMPORTANT To avoid damaging the 61 cm sequencing capillary verify that the entire capillary is secured to the heat plate before closing the heat plate door Close the door over the heat plate With the heat plate door closed check that the capillary has not moved relative to the electrode Resetting the Injection Counte
28. 40 Setting Up DNA Sequencing Experiments About Preparing Extension Products for Electrophoresis About Excess Dye Unincorporated dye terminators must be removed before the samples can be Terminators analyzed by electrophoresis Excess dye terminators in sequencing reactions obscure bases in the early part of the sequence and can interfere with basecalling Precipitation methods are cheaper and faster but they remove less of the unincorporated dye labeled terminators that can obscure data at the beginning of the sequence The spin column procedure removes more terminators but is more costly than precipitation methods For more information refer to the chemistry protocols see page 6 5 Setting Up DNA Sequencing Experiments 6 41 Setting Up Fragment Analysis Experiments Introduction In This Chapter Topics in this chapter include the following Topic See Page Section Setting Up a Fragment Analysis Run 7 3 About Fragment Analysis 7 4 Performing a Fragment Analysis Experiment 7 5 Denaturing and Loading the Samples 7 7 Preparing the Fragment Analysis Sample Sheet 7 9 Preparing the Fragment Analysis Injection List 7 13 Select the Modules Analysis Parameters and Size Standard Files 7 18 Starting the Run 7 20 Section Matrix Files for Fragment Analysis 7 21 About Matrix Files 7 22 Preparing Matrix Standards 7 23 Creating the GeneScan Matrix File 7 25 Setting Up Fragment An
29. 64106608 or 86 10 64106617 86 800 8100497 Hong Kong 852 2756 6928 852 2756 6968 India New Delhi 91 11 653 3743 3744 91 11 653 3138 Korea Seoul 82 2 593 6470 6471 82 2 593 6472 Malaysia Petaling Jaya 60 3 79588268 60 3 79549043 Singapore 65 896 2168 65 896 2147 Taiwan Taipei Hsien 886 2 2358 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 2 532 4484 32 0 2 582 1886 Denmark Naerum 45 45 58 60 00 45 45 58 60 01 Finland Espoo 358 0 9 251 24 250 358 0 9 251 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 Italy Milano 39 0 39 83891 39 0 39 838 9492 Norway Oslo 47 23 12 06 05 47 23 12 05 75 Portugal Lisboa 351 0 22 605 33 14 351 0 22 605 33 15 Spain Tres Cantos 34 0 91 806 1210 34 0 91 806 12 06 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 392400 31 0 180 392409 or 31 0 180 392499 United Kingdom Warrington Cheshire 44 0 1925 825650 44 0 1925 282502 About This Guide 1 11 Region Telephone Fax European Managed Territories EMT Africa English speaking 27 11 478 0411 27 11 47
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31. 9 Actual electrophoresis current The Status window is updated about once per second The log file contains a comprehensive record of all error and status messages generated by the Data Collection software during a run A log file is created for each run and stored in the run folder The log file records Injection parameters Electrophoresis parameters Instrument status at a few different times during data collection for each sample Using the 310 Data Collection Software 4 11 Module used Total number of data points collected for each sample The log file is useful for troubleshooting If there is a problem with the data compare the information recorded in the log file against your expectations for the run Was a parameter set incorrectly Was the wrong module selected Does a component of the instrument fail to reach the set parameters To view the log file select Log from the Window menu The log file is a text file Log log Notepad iol x File Edit Search Help 2 7 01 8 49 19 PH ABI PRISM 316 Data Collection 2 7 801 8 59 19 ABI PRISM 318 Collection version 3 6 6a14 2 7 81 8 49 19 ABI PRISM 318 Firmware version 1 2 2 7 801 8 59 19 Sample Sheet D AppliedBio 316 Sample Sheets Sample Sheet 2 7 801 8 59 19 GeneScan Run Operator 2 7 801 8 59 19 Detector Length 36 cm 2 7 81 8 59 19 Run Started 2 7 81 8 49 26 Injection 1 Fam blue 2 7 81 8 59 28 Module GS STR POP4 1 mL D md4
32. Analysis Parameters Note 50 RFU is the default threshold Rerun sample adding the recommended amount of size standard Lower the value for the size standard color in the Peak Amplitude Threshold field Peaks missing from size standard definition Check the Analysis Parameters in the GeneScan Analysis software to make sure the correct scan range is defined Minimum Peak Half Width is set too high in Analysis Parameters Lower the value for the Minimum Peak Half Width Troubleshooting 8 13 Problems with Peak Number and Position continued Observation Possible Causes Recommended Actions Peak positions off throughout size range fragment analysis Note Refer tothe GeneScan Chemistry Reference for the ABI PRISM 310 Genetic Analyzer for detailed information on factors that affect sizing Incorrect sample sheet Check sample sheet selection in data collection program Change in size calling method Use consistent size calling method Incorrect internal size standard Use correct fragment analysis size standard Incorrect polymer composition Check urea concentration and polymer composition against protocol Incorrect electrophoresis temperature Check the injection list for temperature setting If correct on injection list check the Log for a recording of the actual electrophoresis temperature Incorrectly defined size standard
33. Autosampler GR0980 Parts of the autosampler region Part Purpose Electrode thumbscrew Holds the electrode It also has a hole through which you thread the capillary Cathode electrode Provides a negative pole for electrical current for electrophoresis 48 well tray Holds a maximum of 48 0 5 mL tubes in a 6 x 8 format 96 well tray Holds a maximum of 96 0 2 mL tubes in an 8 x 12 format Autosampler Holds the sample tray and consumable tubes Two 4 mL buffer vials One 1 5 mL Eppendorf tube Upto three 0 5 mL tubes The autosampler moves the tray and tubes so the capillary can be inserted into them Overview of the 310 Genetic Analyzer 2 11 2 12 Overview of the 310 Genetic Analyzer Section Description of the Computer and Software In This Section This section covers the following topics Topic See Page Computer 9 14 Software 2 15 Overview of the 310 Genetic Analyzer 2 13 Computer Overview The computer collects and analyzes data from the 310 Genetic Analyzer System The 310 Genetic Analyzer is shipped with a computer An optional color printer is Requirements available Note Contact technical support for the latest specifications for the computer and the operating system see Technical Support on page 1 9 Note Technical support and field service will support only the computers supplied and configured by Applied Biosystems If you need to rep
34. Dye Module A module file contains the instructions that tell the 310 Genetic 5 Dye Module Analyzer how to run a sample It includes such things as injection time voltage and heat plate temperature Select the default module file that will automatically appear on a newly created injection list Autoanalyze with Analyzes your samples automatically using the analysis program you specify in the drop down list From the drop down list select the software program you want to use to analyze your data 4 24 Using the 310 Data Collection Software GeneScan Injection List Defaults Preferences When you create a new GeneScan injection list and select a sample sheet the software automatically fills out the injection list from the sample sheet You can set how you want the software to fill out the injection list through the injection list preferences Preferences x Sequence Injection List Defaults GeneScan Sample Sheet Defaults Folder Locations File Names Sequence Sample Sheet Defaults GeneScan Injection List Defaults l General Settings Dye Indicators Collection Info Length to Detector fo em Operator 4 Dye Module none M 5 Dye Module none bd Matrix lt none gt M Autoanalyze with D AppliedBio GeneScan Bin AnalyzeGSSample bat Analysis Parameters Analysis Default bd Size Standard none Auto Print OK Cancel For GeneScan injection lists set the following pre
35. ERE 6 38 Thermal Cyclers 4 si xis at eos CER Rhea DIU ai DESI eR NE Ius 6 38 BigDye Terminators lsleeeeeeee I Ih 6 38 Cycle Sequencing Reaction Types 0 0 0 eee eee eee eee 6 39 About Cycle Sequencing ie ous llle a ede wl POS S eS REOR alae ee es 6 40 OOVELVICW toes tte sitet p Set rd abe ios i eas T Snes ating 6 40 Conditions for BigDye Terminators 00 0 eee eee eee 6 40 Cycle Sequencing on the GeneAmp Thermal Cyclers 0 6 40 About Preparing Extension Products for Electrophoresis 00000000000 6 41 About Excess Dye Terminators 0 0 00 eee cece eee eee 6 41 Setting Up Fragment Analysis Experiments Introduction Jio Se ea OO ee i Sate OS a eS Be Stee Se et 7 1 In This Chapter vcs 2 8 st nE uNa he biotin PER ek ool tee E Ee 7 1 About Ls Chapter eec a ated ori teint Ba iva eee ak 7 2 Section Setting Up a Fragment Analysis Run ccce 7 3 In I his SectOne is meu wu VIRESPGLES PINE EUN CE ONSE Pe 7 3 About Fragment Analysis cece cee hmm e 7 4 Types of Experiments i i uude 00th Add Loads iA CAS P aerea e REESE 7 4 Performing a Fragment Analysis Experiment 0 0 0 e eee eee eee 7 5 Materials Required orsi RUE ate te TEE VUES 7 5 Software Required uo wise eae Gates SOA ee EBEN o ER RODEO 7 6 Summary of Procedures 0 eee n 7 6 Denaturing and Loading the Samples 0 0 0 ccc eee eee eee 7 7 About the
36. LEDs 2 5 status indicators 2 4 loading samples fragment analysis 7 8 sequencing 6 10 Log file about 4 11 to 4 12 long term shutdown 3 44 to 3 45 M maintenance autosampler calibrating how to 3 5 calibrating when to 3 4 cleaning how to 3 4 cleaning when to 3 4 homing how to 3 6 homing when to 3 6 buffer replacing when to 3 10 storing 3 10 capillary window cleaning howto 3 14 checking for polymer leaks 3 11 to 3 13 electrode cleaning howto 3 23 cleaning when to 3 23 polymer replacing when to 3 10 resetting the instrument when to 3 46 syringes Index 4 cleaning how to 3 35 cleaning when to 3 35 inspecting 3 37 water replacing when to 3 10 maintenance system See Also computer Manual Control window 4 6 to 4 7 editing a module 4 14 executing a function 4 7 starting a module 4 7 materials required DNA sequencing 6 7 fragment analysis 7 5 Matrix column DNA sequencing 6 12 matrix files 2 20 about 6 24 7 22 creating DNA matrix from a sample 6 30 creating from matrix standards DNA sequencing 6 30 to 6 32 fragment analysis 7 25 to 7 29 errors 6 29 6 31 number of files needed 6 24 7 22 matrix standard samples in sample sheet 7 10 matrix standards checking quality DNA sequencing 6 29 to 6 32 fragment analysis 7 27 to 7 29 denaturing the matrix standard samples fragment analysis 7 24 sequencing 6 26 DNA sequencing part numbers B 4 fragment analysis part numbers B 5 preparing standard samples DN
37. LMS Custom primers for fragment analysis AmpF STR Identifiler PCR Amplification Kit 7 18 Setting Up Fragment Analysis Experiments Size Standard Files Analysis Parameters Files Chemisiry Module Cystic Fibrosis GS STR POP4 1 0 mL C Fragile X Length Polymorphism Assay If you need a custom module see Editing Modules on page 4 14 About Size Standard Files The size standard file holds the results of a run performed with fragments of known length The file can be used to analyze other runs performed under the same conditions to determine the size of fragments of unknown length Applied Biosystems offers several size standard kits Contact your sales representative for more information Generating New Size Standard Files To generate new size standard files set up the instrument and run the mixture of known length fragments as a single sample Create a new size standard file using the GeneScan Analysis software or GeneMapper software See the AB PRISM GeneScan Analysis Software User Guide or the ABI PRISM GeneMapper Software User Guide for more information This file holds the default or custom start and stop point for data analysis the default minimum peak height threshold and the default size calling method New analysis parameters files are created using the GeneScan Analysis software or GeneMapper software See the AB PRISM GeneScan Analysis Software User Guide or
38. Note f you are using collection names you must first enter them in the Collection Info Preferences window see Collection Info Preferences on page 4 28 7 10 Setting Up Fragment Analysis Experiments To create a new fragment analysis sample sheet continued Step Action 8 Click next to the color for the standard the Std column to specify a color for size standard The selected color will be marked with a diamond For 4 dyes this will be red for 5 dyes this will be orange Note Remember the tube arrangement in the tray and the order of the samples on the sample sheet must be the same In the Sample Info column copy the Sample Name information See About the Sample Info Column below 10 In the Comments column enter any additional comments that you want to link to the samples 11 From the File menu select Save As 12 Name the sample sheet and press Return to save it in the Sample Sheets folder About the Sample We recommend copying the sample names into the Sample Info column This Info Column information may be useful in other downstream applications To fill out the Sample Info column Step Action 1 Select the column titled Sample Name From the Edit menu select Copy Select the column titled Sample Info 2 3 4 From the Edit menu select Paste About the Color and Color Std Columns These are the colors of your
39. This Guide 1 2 Safety 1 4 Technical Support 1 9 About This Guide 1 1 About This Guide Overview of Use this table as a quick reference to this guide Contents Chapter Content 1 About This Guide This chapter provides information about the organization of this guide It also provides Safety and Technical Support information Overview of the 310 Genetic Analyzer This chapter describes the parts of the instrument and provides general information about the hardware and software associated with the ABI Prism 310 Genetic Analyzer It also provides a basic explanation of how the 310 Genetic Analyzer works Operating the Instrument Hardware This chapter provides instructions for setting up the 310 Genetic Analyzer for arun It also describes the procedures for operating and maintaining the 310 Genetic Analyzer Using the 310 Data Collection Software This chapter describes the procedures for operating the ABI PRISM9 310 Data Collection Software Injection Lists This chapter describes additional procedures used in preparing injection lists The information in this chapter is meant to supplement the injection list information in Chapters 6 and 7 Setting Up DNA Sequencing Experiments This chapter describes how to set up the 310 Genetic Analyzer for a DNA sequencing experiment Note This chapter provides general information for preparing DNA sequencing exper
40. a Soft Reset 0 0 0 eee eee eee eee e renee 3 47 Performing a Cold Boot ec Goto oh eed pe ees See pee Gee 3 47 Performing a Clear Memory Reset 20 0 00 0 c eee cece eee eee 3 48 About Recovering from a Power Failure 0 0 0 eee eee eee 3 49 Switching Between DNA Sequencing and Fragment Analysis 0 3 50 Switching Between Applications eee eee 3 50 Maintaining and Caring for Your Computer 0 0 0 c eee cee ee eee 3 51 OVELVICW u s cei Ie ee OE ed Bie S E ed Re Big od Ba aed eee Us 3 51 Hard Disk Maintenance css ia n e e eects 3 51 Back Up All Programs and Files Regularly 00 0 0 0 0000005 3 51 Use a Hard Disk Maintenance Program Regularly 00 00 3 51 Use Discretion When Adding Software Programs 000000005 3 51 Monitoring Communication Between the Computer and the Instrument 3 52 About the Communications Diagnostics Window 0 0 2 2 e eee 3 52 Accessing the Communications Diagnostics Window 02 005 3 52 Using the 310 Data Collection Software Introduction 5a aD a Ro RR E T dA Read BS ea 4 In This Chapter eco uRRRDEPR RAI EINER Gl cat PME ES 4 1 Section Using the Data Collection Software ccce eee 4 3 In This Sections eec wLBisG e EbX SE REX UE eae ieee waite 4 3 About the Data Collection Software 0 0 0 cent nnes 4 4 OVERVIEW ies nut E etc ok uox Lc ban
41. added above the injection selected As many lines as are needed can be added in this manner 4GeneScan Injection Sheet Untitled 1 x Sample Sheet SampleSheet042301 9ss v D P Skip Pause Bi Cancel Length to Detector fo cm Operator GS STR POP4 1 mL F md4 Bogus Matrix GS STR POP4 1 mL F md4 i d Bogus Matrix AS three GS STR POP4 1 mL F md4 Bogus Matrix AT four GS STR POP4 1 mL F md4 Bogus Matrix GS STR POP4 1 mL F md4 15 0 i Bogus Matrix Ad one GS STR POP4 1 mL F md4 15 0 Jj Bogus Matrix oe e E Note You can add multiple rows by selecting multiple rows then selecting Insert For example to add two rows above row 4 select rows 4 and 5 then select Insert from the Edit menu 2 To add your new samples click the field in the Tube amp Sample Name column and open the drop down list Select one of your new samples 4GeneScan Injection Sheet Untitled 1 x Sample Sheet SampleSheet042301 9ss v D gt Skip Pause E Cancel Length to Detector fo cm Operator Matrix File Test CCD 4 id 0 A1 one GS STR POP4 1 mL F maa s 150 150 60 24 Bogus Matrix AS two es STR POP4 1 mL F mdg g 5 15 0 15 0 60 24 Bogus Matrix AS three GS STR POP4 1 mL F md4 5 150 150 60 24 Bogus Matrix A four GS STR POP4 1 mL F md4 5 150 150 60 24 Bogus Matrix 41 one
42. 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 to Limited Warranty A 1 What is Conveyed by Purchase A 2 Limited Warranty products or parts furnished by third parties such products or parts will be subject to the warranties if any of their respective manufacturers This Warranty is limited to the original Customer and is not transferable 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 OF ANY OTHER OBLIGATION ON THE PART OF APPLIED BIOSYSTEMS The purchase price of this ABI PRISM 310 Genetic Analyzer includes a grant of license under U S Patent No 5 567 292 and method claims of its foreign counterparts to use this particular instrument for electrophoresis methods employing fluorescence as a means of detection No licenses or rights are conveyed with this purchase either expressly by implication or estoppel Consumables Introduction In This Appendix The following information is included in this appendix Topic See Page Shared Consumables B 2 DNA Sequencing Consumables B 4 Fragment Analysis Consumables B 5 Overview There are specific consumables for use
43. be imported into another 310 Genetic Analyzer s sample sheets or viewed in Microsoft Excel or Word applications 6 14 Setting Up DNA Sequencing Experiments Preparing the DNA Sequencing Injection List Overview The injection list specifies the order for running samples how many injections are made from each sample and the module and running conditions for each injection Preparing an Injection List For a DNA sequencing run you will use the Sequence Injection List From this you can select the Sample sheet Module file For additional information about editing injection lists see Chapter 5 Injection Lists You must fill out an injection list to start a run Note See the Quick Reference Card or Selecting the Module and Dye Set Primer File on page 6 19 for help when filling out the sample sheet and injection list To create an injection list Step Action 1 In the Data Collection software select New from the File menu 2 Select Sequence Injection List Create new x Sequence GeneSean Sequence Sequence GeneScan GeneScan Injection Injection Smpl Sheet Smpl Sheet Smpl Sheet Smpl Sheet List List 48 Tube 96 Tube 48 Tube 96 Tube The Sequencing Injection Sheet window opens Select the sample sheet for the experiment from the Sample Sheet drop down list 4 Sequencing Injection Sheet Untitled 1 x Sample Sheet lt none gt i D P Run Pause
44. compress as the syringe tries to inject polymer into the gel block and capillary The software assumes the syringe travel for compressing the bubble is for polymer delivery More than 15 counts on the syringe drive encoder triggers a Leak Detect error These counts might be the result of compressing a bubble in the syringe Avoid bubbles in the glass syringe when filling it with polymer Checking for Leaks To check for leaks Step Action 1 Open the Data Collection software if not open already 2 From the Window menu select Manual Control 3 Press the Tray button The autosampler tray moves forward 4 Place a small tube containing 0 5 mL of water in tube position 3 in the front of the autosampler 5 Press the Tray button again to move the autosampler back to its original position 6 Select Seq Fill Capillary md4 from the Module drop down list in the Manual Control window Ed Manual Control Ex Function 78 Value Range ceo Pixel Position Y 28 1 to 64 pixels Module nones x a GS Template 1 mL C md4 GS Template 1 mL D md4 P RapidSeq 1mL E md4 P4StdSeq 1mL E md4 Seq Fill Capillary md5 Seq POPE 1 mL A md Seq POPE 1 mL E md4 Seq POP6 Rapid 1 mL A md4 Seq POP6 Rapid 1 mL E md4 v 7 Make note of the position of the end of the glass syringe plunger and write it down Operating the Instrument Hardware 3 11 To check for leaks continued
45. dampened lab wipe PN eh Salt accumulation on the autosampler can cause arcing during electrophoresis Calibrating the When to Calibrate the Autosampler Autosampler The autosampler calibration settings are maintained in the random access memory RAM of the instrument Recalibrate the autosampler After changing the electrode After changing the capillary If the capillary and the septa caps collide After a reset that clears memory 9 9 9 9 When prompted by the instrument An autosampler that is not calibrated can cause damage to the cathode electrode capillary and autosampler and can result in poor data collection or no data due to improper capillary position in a sample tube IMPORTANT The sample tray and Eppendorf tube must be removed before calibrating the autosampler If the sample tray is not removed the electrode will be bent Calibration Points The autosampler tray platform is shown below Use the black or silver dots on the front and rear of the platform to calibrate the autosampler a b Front libration Rear e e cal calibration e 9 point point e 9 3 4 Operating the Instrument Hardware Autosampler Calibration Keyboard Equivalents These are the keyboard equivalents for the autosampler calibration Move to the Key Rear Up Arrow Front Down Arrow Right Right Arrow Left Left Arrow Up Page Up Down Page Down Half Steps Use Shif
46. dialog box cannot be printed However the log file contains the injection list parameters used with the sample and it can be printed 4 14 Using the 310 Data Collection Software About Module The following parameters can be changed only by editing a module Parameters Parameter Description Syringe Pump Time This is the time used to fill the capillary with polymer You may wish to experiment with slightly shorter times to maximize throughput Pre injection Pre injection electrophoresis is a prerun of the refilled capillary Electrophoresis for ion equilibration before samples are electrokinetically injected Using the 310 Data Collection Software 4 15 4 16 Using the 310 Data Collection Software Section Installation and Configuration of the Data Collection Software In This Section This section covers the following topics Topic See Page Installing the Data Collection Software 4 18 Data Collection Software Preference Files 4 19 General Software Procedures 4 29 Using the 310 Data Collection Software 4 17 Installing the Data Collection Software The Data Collection Software Installer Disk Reinstalling the Data Collection Software Installing Modules and Dye Set Primer Files The installer disk is shipped with the instrument The Field Service Engineer installs the 310 Data Collection on the computer when setting up the 310 Genetic Analyzer in your laboratory Keep the Ins
47. dye set used to run the sample If you analyze data with a matrix file that does not reflect the conditions of your run the run will appear to fail Reanalyze with the correct matrix file if you have made this error Create a separate matrix file for each run condition that affects the collection of spectra Some examples are Anew dye is used The pH of polymers or buffers has changed Adifferent type of polymer is used Run the matrix standards under the sample run conditions and make a matrix file Use that matrix file to analyze any and all samples run under those conditions Once a matrix file has been created it can be used for subsequent runs performed With the same kit Onthe same instrument Using the same Modules Set of dyes Polymer After running the matrix standards use their sample files to generate a matrix file using the Sequencing Analysis software 6 24 Setting Up DNA Sequencing Experiments Preparing Matrix Standards About the Matrix Standards An Example Using dRhodamine Matrix Standards You must create a matrix file the first time you use any sequencing method i e dye terminators BigDye terminators dye primers etc The matrix standards can be prepared from matrix standard samples or created directly from a sample or sample standard Preparing matrix standards is optional It is not necessary to use matrix standards to generate a sequencing matrix Any sample that yields goo
48. for DNA sequencing Sequencing Analysis software The Sequencing Analysis software analyzes the raw data and calls the bases It is used for de novo sequencing projects For more information see the AB PRISM DNA Sequencing Analysis Software User Guide SeqScape software The SeqScape software is a fully integrated software tool for a variety of mutation detection applications It is used for comparative sequencing projects For more information see the AB PRISM SeqScape Software for Comparative Sequencing Applications User Guide Fragment Analysis The fragment analysis software enables you to use 310 Genetic Analyzer automated fluorescence detection to size and quantify DNA fragments The program automates the sizing of DNA fragments It also provides flexibility to interactively confirm and fine tune the data analysis and allows you to display the results of an experiment in several different ways The software programs available for analyzing the raw data for fragment analysis GeneScan Analysis software The GeneScan Analysis software analyzes raw data to quantify the DNA fragments and determine the size of the fragments by comparing them to fragments contained in a size standard For more information see the AB PRISM GeneScan Analysis Software User Guide GeneMapper software The GeneMapper software is a high throughput accurate software tool for linkage mapping and human identification applications For more inform
49. not make a matrix You may have designated the wrong files The signal may be too weak to make a matrix The signal may be too high Checking the Matrix Check the quality of the matrix by reviewing the Quality 4 Raw data Analyzed data of the matrix run To check the raw data examine the electropherogram of the raw data For this example the matrix standards should display the following colors Matrix Standard Color in Raw Data dR110 black dR6G green dTAMRA blue dROX red Setting Up DNA Sequencing Experiments 6 29 Creating a Matrix from a Sample Overview t is not necessary to use matrix standards to generate a sequencing matrix Any sample that yields good raw data can be used to create a matrix file The sequencing standard is used to make a matrix in the procedures below The method can be used with other types of samples Reviewing the Data Before making the matrix file review the raw data To review the raw data Step Action 1 View the raw data for all matrix sample files while in the Sample Manager window of the Sequencing Analysis software IMPORTANT Do not analyze the sample files In the Raw Data window verify that the data peaks are present in all four of the standards and that no anomalies are present Note The relative peak amplitude should be less than 4000 y axis of raw data window Note the matrix standard files with the best data for
50. only To fill a syringe Step Action 1 Grasp the syringe by the glass barrel and plunger button 2 Draw 1 0 mL of deionized water slowly into the syringe and then dispense the water completely into a waste container Draw 0 15 mL of polymer slowly into the syringe Raise to eye level and while holding syringe tip up dispense completely into a lint free lab wipe This ensures that the deionized water does not dilute the polymer and if done correctly eliminates bubbles from the syringe Fill the syringe slightly more than desired volume for example if you need 0 5 mL fill to 0 7 mL Raise the syringe to eye level and hold the syringe tip up with graduation scale outlined against a white background Tap the glass barrel with your fingernail to raise air bubbles and then gently push them out using the plunger Move the syringe plunger to the desired volume Blot the syringe tip with a lint free lab wipe without drawing any liquid out of the tip 3 36 Operating the Instrument Hardware To fill a syringe continued Step Action 8 Hold the syringe by the stainless steel hub and screw the filled syringe gently into the gel block finger tight only CAUTION Do not screw syringe into the gel block by holding the glass barrel as this will break the glass barrel Hold only the stainless steel hub Installing the To install the glass syringe S
51. sample and the module and running conditions for each injection For a fragment analysis run you will use the GeneScan Injection List From this you can select the 9 9 9 9 Sample sheet Module file Matrix file Analysis parameters file Size standard file For additional information about editing injection lists see Chapter 5 Injection Lists Preparing an To create a new fragment analysis injection list Injection List Step Action 1 In the Data Collection software go to the File menu and select New The following window appears Create new x ma fa fa Fa Fal Sequence GeneSean Sequence Sequence GeneScan GeneScan Injection Injection Smpl Sheet Smpl Sheet Smpl Sheet Smpl Sheet List List 48 Tube 96 Tube 48 Tube 96 Tube 2 Click GeneScan Injection List Setting Up Fragment Analysis Experiments 7 13 To create a new fragment analysis injection list continued Step Action 3 Select the sample sheet for the experiment from the drop down list This transfers the sample sheet information automatically to the injection list The following is an example of an injection list with matrix standard samples dceneScan Injection Sheet Untitled 2 x Sample Sheet Sample Sheet with Matrix Std g v D gt Run i Pause Cancel Lengthto Detector 30 cm Operator inj Te E tampe Name Module mi secs m Run kv m Run Time Matrix File A 1347
52. that reagents are being stored and used according to manufacturer s instructions Compare with PCR performance using fresh reagents Inconsistent yields with control DNA Combined reagents not spun to bottom of 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 Keep reactions on ice and load immediately Pipetting errors Follow all these precautionary measures Calibrate pipettes Attach tips firmly Check all phases of pipetting technique Whenever possible minimize pipetting small volumes for example make master mixes Note You may also want to consider using a 2 uL or other high precision pipette Troubleshooting 8 21 Problems with Extra Peaks 8 22 Troubleshooting Problems with Extra Peaks Observation Possible Causes Recommended Actions Extra peaks appear with no discernible pattern Mixed sample Verify quality and integrity of sample Presence of exogenous DNA Use appropriate techniques to avoid introducing foreign DNA during laboratory handling Nonspecific priming that is primer template mismatch Check for good primer design Add less template DNA Note High DNA concentrations promote nonspecific annealing Add less primer DNA Note H
53. the AB PRISM GeneMapper Software User Guide for more information Setting Up Fragment Analysis Experiments 7 19 Starting the Run Starting the Run Click the Run button in the Injection List window to start the run Note If you did not preheat the instrument as suggested under Preheating the Heat Plate on page 3 30 it can take up to 30 minutes for the instrument to heat to 60 C and begin electrophoresis Monitoring the Run During the run you can monitor the run and your samples Refer to Run Status and History on page 4 11 for information on monitoring Analyzing Data GeneScan Analysis software automatically analyzes the samples when the run finishes if the automatic analyze checkbox on the injection list is checked Refer to the AB PRISM GeneScan Analysis Software User Guide for more information 7 20 Setting Up Fragment Analysis Experiments Section Matrix Files for Fragment Analysis In This Section This section covers the following topics Topic See Page About Matrix Files 7 22 Preparing Matrix Standards 7 23 Creating the GeneScan Matrix File 7 25 Setting Up Fragment Analysis Experiments 7 21 About Matrix Files Matrix Files How Many Matrix Files About Using Matrix Files Matrix files contain information that corrects for spectral overlap Spectral overlap occurs when part of one dye s emission spectrum falls onto a portion of the detection hardware that is collecting the fluore
54. thermal cycler not manufactured by Applied Biosystems you may need to optimize thermal cycling conditions Ramping time is very important If the thermal ramping time is too fast gt 1 C sec poor noisy data may result For more information refer to the chemistry protocols These protocols are used for the BigDye terminator chemistry These conditions work for a variety of templates and primers However if necessary these parameters can be changed to suit particular situations including the following For short PCR products you can use reduced numbers of cycles e g 20 cycles for a 300 bp fragment Ifthe T of a primer is gt 60 C the annealing step can be eliminated If the T of a primer is 50 C increase the annealing time to 30 seconds or decrease the annealing temperature to 48 C For templates with high GC content 27096 heat the tubes at 98 C for 5 minutes before cycling to help denature the template GeneAmp 9700 9600 or 2400 GeneAmp 9700 9600 or 2400 Step Action 1 Place the tubes in a thermal cycler and set the volume to 20 pL 2 Repeat the following for 25 cycles a Rapid thermal ramp to 96 C 96 C for 10 sec Rapid thermal ramp to 50 C 50 C for 5 sec Rapid thermal ramp to 60 C 60 C for 4 min 000 o0 3 Rapid thermal ramp to 4 C and hold until ready to purify 4 Spin down the contents of the tubes in a microcentrifuge 6
55. using less FJdNTPs fragment analysis Unincorporated F dNTPs fragment analysis Purify the PCR product Unincorporated ddNTPs DNA sequencing Use ethanol precipitation or spin column purification Signal too high in first part of run Excess DNA template depletes nucleotides in reaction mix leading Quantitate template Refer to the sequencing kit protocol for DNA sequencing to excess of short fragments appropriate DNA quantities Peak height Sample evaporation Use septa decreases throughout run High baseline Dirty capillary window Clean capillary window using 95 ethanol and lab wipe Dirty syringe Clean the syringe with warm pure water Dirty pump block Clean the pump block with warm pure water as described in Cleaning the Gel Block on page 3 27 Capillary moved out of position in front of detector window Position capillary in front of laser window Precipitate in polymer Allow polymer to equilibrate to room temperature before using Old defective or incorrectly made buffer or polymer Replace buffer and polymer Dirty detector window Clean the detector window carefully using lense paper Defective capillary Replace the capillary Matrix made incorrectly resulting in too much correction indicated by troughs under peaks Note Matrix problems only show in analyzed data and not the raw data Remake matri
56. will twist and break Always install the capillary on a clean gel block Removing the Capillary from the Packaging Capillaries are shipped in a curved plastic tube Remove a capillary by grasping it between your thumb and forefinger and then pulling and turning the capillary simultaneously Note Be careful not to bend the capillary at the capillary window Connecting the Capillary to the Gel Block To connect the capillary to the gel block Step Action 1 Clean the capillary window with an ethanol dampened lab wipe Open the door covering the heat plate 2 3 Unscrew the plastic capillary fitting partially on the right side of the gel block 4 Screw the capillary fitting partially back into the gel block IMPORTANT Do not tighten the fitting at this point in the procedure or the opening at the tip will be crushed The capillary must be properly inserted through the opening before you tighten the fitting 5 Thread one end of the capillary through the capillary fitting Capillary fitting Waste valve Waste vial Operating the Instrument Hardware 3 15 To connect the capillary to the gel block continued Step Action 6 Adjust the end of the capillary so that it is positioned directly below the opening to the glass syringe It is also acceptable to position the end of the capillary between the intersection of the channels and the capillary fitting IMPORTANT The end of
57. 0 0 0 e 3 6 Filling the Buffer Reservoir eee gd ae Kao ecb ewes ease ee de 3 8 Loadingthe Samples Shiba ae ai ee ee ee heehee 3 9 Butters and Polymer shr 64 Mos ier bus eke Pneu PER ei aa es oak ein EE eee ee 3 10 When to Replace Reagents 2 0 00 0 cee eee eee eee 3 10 Preparing the Polymer and the Electrophoresis Buffer 0 3 10 Making Buffer for a Single Run 0 eee ec eee ene 3 10 Storing the Butler osteitis ae eh at oe ieee iy RUNE VU e PUE 3 10 Checking for Polymer Leaks 0 ea a ea a T eens 3 11 Capillary eet tec tec pcc goa etc e ae ah eve ae ae 3 14 About the Capillary 3 24 cbe data ee A Eu e Los 3 14 Cutting the Capillary 060 54s2 0 beet DES DIS es BAN EXE PERPE DE nts 3 14 Cleaning the Capillary Window 0 0 00 cece cee eee ee 3 14 Installing the Capillary 2 2 It RE eR pe re EUR 3 15 Testing the Capillary Window lsseleeeeeeeee III 3 19 Removing and Storing the Capillary 0 0 00 eee eee eee 3 20 Verifying that the Capillary Fills Correctly 0 00 eee eee eee eee 3 22 Avoiding Capillary Clogs ssi scote Lok ee had we RE RR oly be ee es 3 22 Electr de doce Sue a ee amy a a ee eke eens eov ttc 3 23 OVetVIeW s as eee see EUST ERI I oR teas en e edes 3 23 Cleaning the Electrode psss 2e eg e RE RR ex Re ES 3 23 Installing the Electrode tatti eui conei to teei pa ieni B 3 24 Trimming the Electrod 2 0 0 ee neser ur eup eh 3 25 Straightening
58. 0 0 30 Boge Test CCD 4 Color md4 X 7 j X Bogus Matrix z GS STR POP4 1 mL F md4 Bogus Matrix E STR POP4 1 mL F md4 5 24 Bogus Matrix S STR POP4 1 mL F md4 i ij Bogus Matrix S STR POP4 1 mL F md4 i i Bogus Matrix bs STR POP4 1 mL F md4 Bogus Matrix Injection Lists 5 7 To change the order of samples during a run continued Step Action 3 Continue to change the selected samples in the Tube amp Sample Name column until you have the order you want 4GeneScan Injection Sheet Untitled 1 x Sample Sheet Sample Sheet042301 0ss v D gt Skip Pause Bi Cancel Length to Detector fo cm Operator GSSTRPOP4 1mL Fimd4 5 15 0 amp 0 24 Bogus Matrix GS STR POP4 1 mL F md4 Bogus Matrix GS STR POP4 1 mL F md4 i f Bogus Matrix GS STR POP4 1 mL F md4 i jJ Bogus Matrix A11 six GS STR POP4 1 mL F md4 Bogus Matrix ae e Rows in the injection list can also be cut from one location and pasted into another Holding down the option key when selecting an injection number selects the whole row To cut and paste rows within an injection list Step Action 1 Select the row you want to move 2 From the Edit menu select Cut 3 Select the row below where you want to move the row 4 From the Edit menu select Paste 5 8 Injection Lists
59. 1 A1 BDT Std E 2 A3 SeqStd GS Template 1 mL C md4 3 A1 BDTStd IGS Template 1 mL D md4 3 P4RapidSeq 1mL E md4 P4StdSeq 1mL E md4 Seq Fill Capillary md4 seq POPB 1 mL A md4 Seq POPS Rapid 1 mL amp md4 Select the module for the sample In the Modules column hold down the Shift key and select the fields for all the subsequent samples that require that module Sequencing Injection Sheet Untitled 1 x Sample Sheet 22701 kksss x D P Run Pause Wi Cancel Length to Detector fo cm Operator Ini Tube amp Sample Name Inj Secs Inj kV Run kV Run C Run Time Finish Note To select the entire column click the column header 5 4 Injection Lists Changing the Module for a Sample Making Multiple Injections from One Sample Tube To use the fill down function continued Step Action 4 Select Fill Down from the Edit menu All selected Module fields will be filled in with the same module Sequencing Injection Sheet Untitled 1 Ex Sample Sheet 022701 kk sss hd D P Run Pause E Cancel Length to Detector fo cm Operator Inj Tube amp Sample Name Inj Secs Inj kV Run kV Run C Run Time Finish OPG 1 mL E md4 Aj P6 1 mL E md4 1 mL E md4 B 1 mL E md4 OP6 1 mL E md4
60. 10 Genetic Analyzer Site Preparation and Safety 903558 Guide ABI PRISM DNA Sequencing Analysis Software User Guide 4308924 Automated DNA Sequencing Chemistry Guide 4305080 ABI PRISM SeqScape Software for Comparative Sequencing 4326982 Applications User Guide ABI PRISM GeneScan Analysis Software User Guide 4308923 ABI PRISM GeneMapper Software User Guide 4318910 GeneScan Chemistry Reference for the ABI PRISM 310 Genetic 4303189 Analyzer Comparative PCR Sequencing a Guide to Sequencing Based Stock No 770901 001 Mutation Detection Guide to PCR Enzymes Stock No 700905 POP 4 Polymer Sequencing Protocols for the 310 Genetic 4327927 Analyzer User Bulletin About This Guide 1 3 Safety Documentation User Attention Words Chemical Hazard Warning 1 4 About This Guide Five user attention words appear in the text of all Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Calls attention to useful information IMPORTANT Indicates information that is necessary for proper instrument operation y Ner Vue Indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WIE Indicates a potentially hazardous situation which if not avoided could result in death or serious injury 7 NA eldi Indicates an imminently hazardous situation which if not avo
61. 10X Genetic Analyzer Buffer with EDTA to a 1X concentration 50 mL total volume with distilled deionized water Note The 10X Genetic Analyzer Buffer with EDTA can be diluted in a smaller volume Diluted buffer can be stored at 4 C and used within 2 weeks Making Buffer for a You can prepare fresh electrophoretic buffer for a single run by combining Single Run 13 mL of 10X Genetic Analyzer Buffer with EDTA 11 7 mL deionized water You can also prepare a stock 1X solution of analysis buffer by combining 5mL 10X Genetic Analysis Buffer with EDTA 45 mL deionized water Store the stock buffer at 2 to 8 C Storing the Buffer Store the electrophoresis buffer at 2 to 8 C The 1X buffer can be stored for 2 weeks 3 10 Operating the Instrument Hardware Checking for Overview Polymer Leaks cas can cause high rates of polymer consumption Severe leaks usually mean one or more of the manual valves are not closed Leaks may also occur at the ferrule sealing the capillary to the gel block at the Teflon seal of the plunger in the glass syringe or at the valve to the buffer reservoir Check the base of the glass syringe for polymer leaks visible as a white residue especially at the interface between the glass barrel and the metal tip About the Leak Detect Error If the instrument pauses with a Leak Detect error message check for Polymer leaks Bubbles in the glass syringe Bubbles in the glass syringe will
62. 2 0 0 cece ee nee 3 39 Recommendations di db RR pO RCo a hada Me RR Re ERU R EUR 3 39 Section General Instrument and Computer Procedures eese 3 41 In This Section eriei eire ewe gine eb sites be ae a ote PER SU ER ESPACES OS 3 41 General Cares iss care BP EE OR UR Me Sr Soe cas 3 42 Laboratory Temperature vse cios eesi e A a Ea Ie 3 42 Laboratory Humidity 0 0 eee eee eee 3 42 Voltage S pIK68 5 e eee ee eO Aenea Mee co A aad dee eue cals es 3 42 Cle ning neospoeREPRUEOPeUESeeriE esRpkeeBiWe0IleRGGeESeeeDse pEPROES 3 42 Cables 5e ap DOG AN ase ie d E e ee LEUR MUR EUR 3 42 Turning On the Genetic Analyzer sleseeeeeeeeee eens 3 43 If a Capillary Is Already Installed lees 3 43 Turning On the Instrument 2 0 0 0 ee 3 43 Shutting Down the Genetic Analyzer sseseeeeeeeeeee ee 3 44 About a Short Term Shutdown lesen 3 44 Performing a Short Term Shutdown eeeeleeeeeeeeeeeeen 3 44 About a Long Term Shutdown leseeeeeeeeee eee 3 44 Performing a Long Term Shutdown 0 00 0 0 cece eee ee eee 3 45 Resetting the Genetic Analyzer 0 0 0 cece eee ene ee 3 46 About Firmware sss sane Se salep sient es Pe alee EE Melba s fed HUET Hee 3 46 Types OF Resets oot oso Pe Ub gt EB Kar rebr est EAS em 3 46 WEI tO Reset ire ch bleed eke sec beg hb bodega eects bd cece bred ect merde 3 46 Troubleshooting with Resets llle II 3 46 Performing
63. 20 GeneMapper software 2 15 2 16 RAM requirements 2 14 General Settings preferences 4 26 GeneScan Analysis Software 2 15 2 16 RAM requirements 2 14 GeneScan Injection List Defaults 4 25 GeneScan Sample Sheet Defaults 4 23 GeneScan sample sheet examples 7 10 Global Serial Number using to automatically number files 4 26 green LED light status defined 2 4 H hard disk maintenance 3 51 history 4 11 to 4 13 Electrophoresis History window 4 13 Log file about 4 11 to 4 12 homing autosampler 3 6 syringes 3 38 humidity in the laboratory 3 42 I injection list adding new samples to injection list 5 10 adding rows 5 6 adding samples to instrument 5 11 adding samples to sample sheet 5 9 changing run parameters 5 7 Index 3 changing sample order 5 7 changing the module forasample 5 5 cutting and pasting rows 5 8 fill down function 5 4 GeneScan defaults preferences 4 25 how to fill out DNA sequencing injection list 6 15 to 6 16 fragment analysis injection list 7 13 making multiple injections 5 5 preferences 4 24 to 4 26 preparing DNA sequencing 6 15 to 6 17 fragment analysis 7 13 to 7 16 Sequencing defaults preferences 4 24 installing capillary on empty gel block 3 15 to 3 19 Cathode electrode 3 24 dye set primer files 4 18 Internet address customer training information 1 13 Documents on Demand 1 12 L Leak Detect error 3 11 leaks polymer checking for 3 11 to 3 13 syringe correcting leaks at 3 12 LEDs lights service
64. 20 to 3 21 how to store capillary off the instrument 3 20 how to store capillary on the instrument 3 20 summary of procedures fragment analysis 7 6 Syringe Max Travel setting 3 37 syringes basic components 3 35 cleaning 3 35 correcting leaks at the syringe 3 12 filling 3 36 handling and storing 3 39 homing 3 38 inspecting 3 37 installing 3 37 overview 3 35 pump time editing 4 15 recommendations 3 39 removing 3 37 setting Syringe Max Travel 3 37 warranty information 3 35 system requirements 2 14 T technical support 1 9 to 1 13 e mail address 1 9 Internet address 1 12 regional sales offices 1 11 to 1 12 telephone fax North America 1 9 1 11 temperature electrophoresis DNA sequencing 6 18 fragment analysis 7 17 temperature of the laboratory 3 42 Template Suppression Reagent TSR 6 9 templates characteristics of poor template preparation 6 35 training obtaining information 1 13 trays See sample trays trimming Cathode electrode 3 25 troubleshooting amplification problems with 8 18 to 8 21 automatic data analysis problems with 8 5 current problems with 8 6 to 8 7 peaks problems with 8 12 to 8 14 8 16 to 8 17 8 22 to 8 23 polymer problems with 8 4 primer problems with 8 3 signal strength and quality problems with 8 8 to 8 11 troubleshooting howto 8 2 TSR sample mix preparing 6 9 tubes troubleshooting not seated tightly 8 19 sample not at bottom 8 8 wrong PCR tube 8 19 V virtual filters 2 20 to 2 21 and
65. 4 15 Q quick reference for this guide 1 2 Quick Reference Card 1 3 quickstart experiments 6 30 DNA sequencing cycle sequencing using terminators 6 5 injection list preparing 6 15 to 6 17 materials required 6 7 matrix file creating 6 30 to 6 32 matrix standards preparing 6 25 samples denaturing and loading 6 9 to 6 10 summary of procedures 6 7 fragment analysis 7 1 to 7 20 injection list preparing 7 13 to 7 16 materials required 7 5 matrix file creating 7 25 to 7 29 samples denaturing and loading 7 7 to 7 8 summary of procedures 7 6 quickstart preparing the instrument 3 2 quitting 3 44 to 3 45 R RAM requirements 2 14 raw data and analyzed data difference between 4 9 Raw Data window 4 8 color buttons 4 9 raw data and analyzed data difference 4 9 resizing 4 8 reaction mixture preparing a portion 6 10 reagents 6 38 buffer 3 10 polymer 3 10 preparing 3 10 sample preparation 7 7 when to replace 3 10 red LED light status defined 2 4 reference table run modules and dye set primer files 6 19 removing capillary for storage 3 21 capillary from packaging 3 15 syringe 3 37 reset button back of instrument 2 6 resetting 310 Genetic Analyzer 3 46 to 3 48 clear memory reset performing 3 48 clear memory reset recording calibration data 3 48 cold boot performing 3 47 factory settings 4 27 firmware about 3 46 soft reset performing 3 47 troubleshooting with resets 3 46 types of resets 3 46 when to reset 3 46 run modu
66. 6 10 summary of procedures 6 7 fragment analysis 7 1 to 7 20 analyzing data 7 20 injection list preparing 7 13 to 7 16 materials required 7 5 matrix file creating 7 25 to 7 29 samples denaturing and loading 7 7 to 7 8 F Field Service in North America contacting 1 9 File Location preferences dialog box 4 20 File Names preferences dialog box 4 21 files autonumbering 4 26 location how to find 4 29 firmware about 3 46 resending 3 47 formamide 7 7 formamide sample mix 7 7 diluting 7 8 formamide size standard 7 7 fragment analysis 7 1 to 7 20 about 2 18 application kits part numbers B 5 color guide for displaying raw data 2 22 consumables B 5 consumables part numbers B 6 injection list how to fill out 7 13 materials required 7 5 matrix files creating fragment analysis matrix file 7 25 to 7 29 matrix standards part numbers B 5 modules 7 18 preparing a sample sheet 7 9 preparing matrix standard samples 7 23 size standards part numbers B 5 summary of procedures 7 6 switching to from DNA sequencing appplication 3 50 types of experiments 7 4 virtual filters 4 10 fragmented memory using hard disk maintenance program 3 51 G gel block installing capillary on empty block 3 15 to 3 19 after installing 3 19 connecting to block 3 15 to 3 16 positioning in the detector 3 17 positioning near the electrode 3 18 resetting injection counter 3 18 gel block region parts of 2 8 to 2 9 gels evaluating samples using an agarose gel 8
67. 8 0349 Johannesburg South Africa Africa French speaking 33 1 69 59 85 11 33 1 69 59 85 00 Paris France India New Delhi 91 11 653 3743 91 11 653 3138 91 11 653 3744 Poland Lithuania Latvia and Estonia 48 22 866 40 10 48 22 866 40 20 Warszawa For all other EMT countries not listed 44 1925 282481 44 1925 282509 Central and southeast Europe CIS Middle East and West Asia Japan Japan Hacchobori Chuo Ku Tokyo 81 3 5566 6230 81 3 5566 6507 Latin America Caribbean countries Mexico and 52 55 35 3610 52 55 66 2308 Central America Brazil 0 800 704 9004 or 55 11 5070 9694 95 55 11 5070 9654 Argentina 800 666 0096 55 11 5070 9694 95 Chile 1230 020 9102 55 11 5070 9694 95 Uruguay 0004 055 654 55 11 5070 9694 95 To Reach Technical Support Through To contact Technical Support through the Applied Biosystems web site the Applied Step Action Biosystems Web Site SEE 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page then click Frequently Asked Questions 3 Click Contact Support in the contents list at the left of the screen Click your geographic region for the product area of interest 5 In the Personal Assistance form enter the requested information and your question then click Ask Us RIGHT NOW 6 In the Customer Information form enter the requested information then click Ask Us RIGHT NOW Within 24 to 48 h
68. 9 Updated Files 5 5 ooi ce deen Bae oe hada Mine RE ERR 6 19 MOdUles asa nates a eas coed Spanier aan E tat pe asia hoes ta utah xg 6 19 Mobility Set Primer Filesi inneren cc eens 6 19 Reference Table for Dye Set Primer Files and Modules 6 19 Starting the Run secsi ri tenenti MERDA E pU RPENEP dae PS UN e a 6 22 Startngthe RUM 3 0008 sed free aes Gad oleae HERS Soke UP OPUS CUEDEDES 6 22 Monitoring the Run 00 6 yee rms EH ETE ete 6 22 Analyzing the Data eee e A e nA pP Cao beeen Sa be abe 6 22 Section Matrix Files for DNA Sequencing celere 6 23 In This Section vos ee hie hee tetas TEMERE 6 23 About Matrix Biles ce ose oc aoe SU a ee ant SU a ae se Si E 6 24 Matrix Files 4o pu CR de ah od shad Ms M RRCRA ON Ege teg ess 6 24 How Many Matrix Files lleslsleleeeeee ee 6 24 About Using Matrix Files 0 II 6 24 Preparing Matrix Standards 00 0 cee enn ee 6 25 About the Matrix Standards llle eee 6 25 An Example Using dRhodamine Matrix Standards 0 0 6 25 Preparing Matrix Standard Samples 0 0 00 6 26 Denaturing the Matrix Standard Samples slsleleleeee eee eee 6 26 Creating Matrix Files from Matrix Standards 0 0 00 cece eee eee 6 27 About This Example srice octo pA ADR Er urge SR ae Tee 6 27 Reviewing the Data ek es Me eat Sh RN Re NR eRe wwe Eine 6 27 Makijs a Matnx File 4 24s sesh ici ana ea a ton 6 27 If Yo
69. A sequencing 6 25 fragment analysis 7 23 mobility files See dye set primer files modules editing 4 14 installing 4 18 pausing 4 7 selecting DNA sequencing 6 19 fragment analysis 7 18 starting 4 7 monitoring raw data 4 8 run of the instrument DNA sequencing 6 22 fragment analysis 7 20 O On Off switch 2 6 P parameters voltage and time DNA sequencing 6 17 fragment analysis 7 17 Pause Alert window using when leak detector triggered 3 21 pausing arun 4 7 peaks troubleshooting extra peaks 8 22 to 8 23 number and position 8 12 to 8 14 quality and resolution 8 16 to 8 17 polymer 3 10 bubbles in 3 11 checking for leaks 3 11 to 3 13 correcting leaks at the buffer reservoir 3 12 correcting leaks at the syringe 3 12 DNA sequencing polymer types available B 4 fragment analysis types available B 5 preparing 3 10 replacing when to 3 10 polymer not enough 8 4 polymer troubleshooting 8 4 power cord about 2 6 power cords and cables routing 3 42 power down warning 3 49 power failure recovering from 3 49 power troubleshooting 8 6 to 8 7 preferences 4 19 to 4 27 Dye Indicators preferences 4 27 File Location preferences 4 20 File Names preferences 4 21 General Settings preferences 4 26 Injection List preferences 4 24 to 4 26 sample sheets preferences 4 22 to 4 23 pre injection electrophoresis editing parameter 4 15 primer design 6 37 primer files choosing for sequencing 6 19 primer troubleshooting 8 3 pump time syringe
70. ABI PRISM 310 Genetic Analyzer User Guide gt Applied KS Biosystems Copyright 2001 2010 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document This instrument Serial No is Authorized for use in DNA sequencing and fragment analysis This authorization is included in the purchase price of this instrument and corresponds to the up front fee component of a license under process claims of U S patents 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 Authorization 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 li
71. D Red dROX PET ROX ROX TAMRA ROX Orange LIZ LIZ 2 22 Overview of the 310 Genetic Analyzer Operating the Instrument Hardware Introduction In This Chapter Topics in this chapter include the following Topic See Page Preparing the 310 Genetic Analyzer for a Run 3 2 Section Procedures for Setting Up and Maintaining the Instrument 3 3 Autosampler 3 4 Buffers and Polymer 3 10 Capillary 3 14 Electrode 3 23 Gel Block 3 27 Heat Plate 3 30 Sample Trays 3 31 Syringes 3 35 Section General Instrument and Computer Procedures 3 41 General Care 3 42 Turning On the Genetic Analyzer 3 43 Shutting Down the Genetic Analyzer 3 44 Resetting the Genetic Analyzer 3 46 Switching Between DNA Sequencing and Fragment Analysis 3 50 Maintaining and Caring for Your Computer 3 51 Monitoring Communication Between the Computer and the Instrument 3 52 Operating the Instrument Hardware 3 1 Preparing the 310 Genetic Analyzer for a Run Summary of The following table lists in order the procedures used when setting up the ABI PRISM9 Procedures 310 Genetic Analyzer for a run Once you are familiar with the instrument and its operation you can refer to the Quick Reference Card to get a run started quickly Before using the instrument make sure you are familiar with the safety information in Chapter 1 About This Guide Prepare the 310 Genetic Analyzer by
72. Define size standard peak sizes separately for each incorrectly sized injection Runs get progressively slower peaks come off at higher and higher scan numbers Capillary not refilling a Check for leaks b Check for bubbles in the syringe c Replace capillary if clogged d Increase capillary refill time Syringe out of polymer Fill syringe with fresh polymer Ambient temperature in laboratory changing Ensure that the ambient temperature is between 15 and 30 C whenever the instrument is in operation including nights and weekends Do not keep the instrument in direct sunlight Runs get progressively faster peaks come off at lower and lower scan numbers Water in syringe When loading syringe a Prime syringe with small volume of polymer b Invert syringe to coat capillary walls c Discard polymer d Fill syringe with fresh running polymer Ambient temperature in laboratory changing Ensure that the ambient temperature is between 15 and 30 C whenever the instrument is in operation including nights and weekends Do not keep the instrument in direct sunlight 8 14 Troubleshooting Problems with Peak Number and Position continued Observation Possible Causes Recommended Actions Peak spacing too high DNA sequencing Capillary clog See Avoiding Capillary Clogs on page 3 22 Arcing to conductive surface on the inst
73. Preparing Matrix Standards 6 25 Creating Matrix Files from Matrix Standards 6 27 Creating a Matrix from a Sample 6 30 Section Useful Information for Preparing DNA Sequencing Samples 6 33 About DNA Template Preparation 6 34 About DNA Template Quality 6 35 About Primer Design 6 37 About Preparing Sequencing Reactions 6 38 About Cycle Sequencing 6 40 About Preparing Extension Products for Electrophoresis 6 41 Setting Up DNA Sequencing Experiments 6 1 About this Chapter This chapter summarizes the steps for using the ABI PRISM9 310 Genetic Analyzer to run a typical DNA sequencing experiment This chapter explains the steps for making a matrix file from Matrix standards from multiple samples Asingle four color sample If you are familiar with operating the 310 Genetic Analyzer you can quickly set up a sequencing run by following the instructions on the Quick Reference Card For detailed information about setting up and operating the 310 Genetic Analyzer see Chapter 3 Operating the Instrument Hardware 6 2 Setting Up DNA Sequencing Experiments Section Setting Up a DNA Sequencing Run In This Section This section covers the following topics Topic See Page About Sequencing Analysis 6 4 Performing a DNA Sequencing Experiment 6 7 Denaturing and Loading Samples 6 9 Preparing the DNA Sequencing Sample Sheet 6 11 Preparing the DNA Sequencing Injection List 6 15 Selecting the Module and Dye Set Prim
74. Sample Preparation Reagents 0 0 00 cece eee eee 7 7 About Formamide and Samples in Formamide 0 00 00 0c e eee eee 7 7 Preparing the Formamide Sample Mix 0 0 0 eee eee eee eee ee 7 7 Diluting the Formamide Sample Mix 0 0 0 0 eee eee eee 7 8 Minimum Sample Volume 00 0 e eee eee eens 7 8 Loading the Sampl s o esee Githa a dy Ree RR Sb ERR dex PS RES 7 8 Preparing the Fragment Analysis Sample Sheet 0 0 0 0 00 eee ee eee 7 9 About Sample Sheets 0 0 eee ce ee e In 7 9 Preparing a Sample Sheet 0 0 ee eens 7 9 About the Sample Info Column 0 0 20 eee eee 7 11 About the Color and Std Columns 0 00 0 0 e eee eee 7 11 Using a Previously Created Sample Sheet 0 00000 e eee eee 7 12 Importing Sample Sheets 0 0 cee eee 7 12 Exporting Sample Sheets secese teesin sono ce ee eee 7 12 Preparing the Fragment Analysis Injection List 0 0 0 0 0 eee eee eee eee 7 13 OMERVICW since sesto haer bui ohbsutebuss cite mee cas oscar 7 13 Preparing an Injection Lists sssi sees nun errue creua cee eee 7 13 GeneScan Injection List Fields 2 0 0 0 00 cece eee 7 15 Editing Injection Lists 0 0 0 cc cee eee ee 7 16 About Electrokinetic Injection Voltage and Time 0 0 e ee eee eee 7 17 Electrophoresis Running Voltage for Fragment Analysis lees esses 7 17 Electrophoresi
75. Section General Instrument and Computer Procedures In This Section This section covers the following topics Topic See Page General Care 3 42 Turning On the Genetic Analyzer 3 43 Shutting Down the Genetic Analyzer 3 44 Resetting the Genetic Analyzer 3 46 Switching Between DNA Sequencing and Fragment Analysis 3 50 Maintaining and Caring for Your Computer 3 51 Monitoring Communication Between the Computer and the Instrument 3 52 Operating the Instrument Hardware 3 41 General Care Laboratory Temperature Laboratory Humidity Voltage Spikes Cleaning Cables Ensure that the ambient temperature is maintained between 15 and 30 C 59 and 86 F whenever the instrument is in operation including nights and weekends Once the instrument is set up and in operation the laboratory temperature should not fluctuate more than 2 C Do not keep the instrument in direct sunlight or under heating or air conditioning vents Ensure that the humidity is below 80 Avoid excessive condensation on the instrument Do not plug the computer and 310 Genetic Analyzer into the same circuit used by other laboratory devices especially centrifuges refrigerators or freezers Ensure that the circuit is grounded Check with your laboratory s building facilities personnel if you have questions about the quality of power in your laboratory Put the computer and 310 Genetic Analyzer on a line conditioner or an u
76. See Positioning the Capillary in the Detector on page 3 17 Volume of sample too low Sample volume must be 10 pL Samples added to formamide that has degraded to formic acid and formate ions leading to injection of insufficient sample Use freshly deionized formamide See Denaturing and Loading Samples on page 6 9 or page 7 7 Salts in sample leading to injection of insufficient sample Remove ions by Dialyzing sample Ethanol precipitation Spin column purification Sample not thoroughly mixed Mix sample by pipetting up and down several times Template not accurately quantitated DNA sequencing Quantitate template Refer to the sequencing kit protocol for appropriate DNA quantities Insufficient FJdNTPs added to PCR reaction fragment analysis Reamplify using more FJdNTPs or examine the efficiency of the PCR Capillary and or electrode not immersed in sample Recalibrate the autosampler Insufficient injection time Increase the electrokinetic injection time or voltage CCD camera not calibrated Contact your service representative Troubleshooting 8 9 8 10 Troubleshooting Problems with Signal Strength and Quality continued Observation Possible Causes Recommended Actions Signal too high Too much sample injected into capillary Decrease injection time or injection voltage Dilute sample Reamplify
77. VING PARTS Syringe guide Gel pump button Tray button Anode buffer valve actuator Anode buffer valve Anode electrode Anode buffer reservoir Drip tray Parts of the gel block Glass syringe Luer valve Capillary fitting Waste valve Waste vial Part Purpose Syringe drive Provides positive pressure to the syringe Syringe drive toggle Allows you to switch the syringe drive to either the glass or plastic syringe Syringe guide Holds the plastic and glass syringes in position Gel pump button This button is not used for normal operation of the instrument at this time Tray button Moves the autosampler in and out so you can put a sample tray on the autosampler or a sample into the sample tray Anode buffer valve actuator Opens and closes the buffer valve 2 8 Overview of the 310 Genetic Analyzer Parts of the gel block continued Part Purpose Anode buffer valve Closes access to the buffer when filling the capillary Opens access to the buffer for electrophoresis Anode electrode Provides a positive electrical current for electrophoresis Anode buffer reservoir Contains the buffer required for electrophoresis Drip tray Catches any liquids that drip from the gel block Glass syringe Stores the polymer between runs and generates the necessary forc
78. Wi cancel none Length to DetectiOther 022701 kk sss Inj Tube amp SiSample Sheet TEST sss Inj Secs inj kv Run kv Run C Run Time Finish Time ri srl Setting Up DNA Sequencing Experiments 6 15 To create an injection list continued Step Action 4 The injection list is automatically filled in with information from the selected Sample Sheet 5 Fill in the module information from the Module drop down list See Selecting the Module and Dye Set Primer File on page 6 19 if you need help selecting the correct file Sequencing Injection Sheet Untitled 1 x Sample Sheet 22701 kksss x D P Run Pause E Cancel Length to Detector fo cm Operator Module Inj Secs Inj kV Run kV Run C Run Time Finish nones E a GS Template 1 mL C md4 GS Template 1 mL D md4 1 P4RapidSeq 1mL E md4 P4StdSeq 1mL E md4 Seq Fill Capillary md4 Seq POPS 1 mL A md4 Seq POPS Rapid 1 mL A md4 w Note Drag the column markers at the top of columns to change their width Fields in the sequencing injection list form Field Description Sample Sheet Displays the selected sample sheet name Page Icon Opens the selected sample sheet Length to Detector The distance between the end of the capillary that is in the sample and the capillary window The length you enter wil
79. aks are present in each sample Peak data should be on scale and the dye of interest should have a value between 200 to 2000 RFU Check for any data anomalies such as an unstable baseline Rerun samples that have an unstable baseline Select a starting point for the matrix data The starting point for matrix data should be slightly beyond the point where the primer peak falls back to the baseline Make sure you have collected a sufficient number of peaks A typical fragment analysis run yields a minimum of seven peaks Setting Up Fragment Analysis Experiments 7 25 Making a Matrix To make a matrix file File Step Action 1 From the File menu select New an x Create New SL BE 3 l Project Foalysis Size Matrix Parameters Standard Click the Matrix icon The Make New Matrix dialog box opens il Make New Matrix Select the Matrix Standard Sample Files Number Of Dyes 5v B No File Selected for B Data Start At o G No File Selected for G Data Start At o n No File Selected for Y Data Start At o R No File Selected for R Data Start At o oO No File Selected for D Data Start At o Points 00000 Cancel Gr Note The example above shows a five dye set Click the B G Y R and O buttons to choose the standard sample files Choose the sample file representing blue dye for B green dye for G orange dye for O etc E
80. alysis Experiments 7 1 About This Chapter This chapter summarizes the steps for using the ABI PRISM 310 Genetic Analyzer to run a typical fragment analysis run with pooled DNA This chapter assumes you have prepared the 310 Genetic Analyzer as described in Preparing the 310 Genetic Analyzer for a Run on page 3 2 For more information about operating the instrument refer to Chapter 3 Operating the Instrument Hardware 7 2 Setting Up Fragment Analysis Experiments Section Setting Up a Fragment Analysis Run In This Section This section covers the following topics Topic See Page About Fragment Analysis 7 4 Performing a Fragment Analysis Experiment 7 5 Denaturing and Loading the Samples 7 7 Preparing the Fragment Analysis Sample Sheet 7 9 Preparing the Fragment Analysis Injection List 7 13 Select the Modules Analysis Parameters and Size Standard Files 7 18 Starting the Run 7 20 Setting Up Fragment Analysis Experiments 7 3 About Fragment Analysis Types of There are many types of experiments that are analyzed using the 310 Genetic Experiments Analyzer and the ABI PRISM GeneScan Analysis Software or ABI PRISM GeneMapper Software They all attempt to determine the size or relative quantity of DNA fragments Applied Biosystems sells a number of kits for applications that are analyzed with GeneScan Analysis software or GeneMapper software Contact your sales representative for informatio
81. atic Data Analysis Problems 8 5 Current Problems 8 6 Signal Strength and Quality Problems 8 8 Peak Number and Position Problems 8 12 Problems with Peak Quality and Resolution 8 16 Problems with Poor Amplification 8 18 Problems with Extra Peaks 8 22 Troubleshooting 8 1 How to Troubleshoot Data Troubleshooting To troubleshoot ABI PRISM 310 Genetic Analyzer problems Data Step Action 1 Understand The chemistry Labeling of the samples How the 310 Genetic Analyzer collects data How the data analysis software programs work Review the experiment for errors in primer design sample quantitation and purification pipetting problems software preference settings and other common mistakes 2 Examine the data Describe the problem as specifically as possible Isit a problem with the sample peaks the baseline or the peaks of only one color Does the problem exist in all parts of the run or does it affect only DNA fragments of a certain length Isthe problem visible in raw data analyzed data log files Continue to ask these types of questions until you have described the problem as specifically and thoroughly as possible 3 List possible causes of the problem See the troubleshooting tables in this chapter for help 4 For each possible cause you have listed ask Does other information support or contradict this as the cause of the problem 5 If necessary collect more inf
82. ation keyboard equivalents autosampler calibration points autosampler 3 4 calibration test autosampler 3 6 cancelling running module 4 7 capillary 3 14 to 3 22 cleaning the capillary window 3 14 cutting 3 14 installing on empty gel block 3 15 to 3 19 after installing 3 19 connecting to block 3 15 to 3 16 positioning in the detector 3 17 positioning near the electrode 3 18 resetting injection counter 3 18 part numbers B 2 reinstalling a stored capillary 3 21 removing for storage 3 21 removing from packaging 3 15 setting time to refill capillary 4 15 storing 3 20 to 3 21 3 5 Index 1 storing off the instrument 3 20 storing on the instrument 3 20 testing position of window 3 19 verifying correct fill 3 22 capillary clogs how to avoid 3 22 capillary window cleaning 3 14 testing position 3 19 Cathode electrode preparing 3 23 to 3 26 cleaning 3 23 installing 3 24 straightening 3 26 trimming 3 25 cleaning syringes 3 35 cleaning general recommendations 3 42 clear memory reset performing 3 48 reset recording calibration data 3 48 clogged capillary how to avoid 3 22 cold boot performing 3 47 Collection Name column DNA sequencing 6 13 fragment analysis 7 10 color assigned to bases after data analysis 4 9 guide raw data displays 2 21 color buttons 4 9 Color column fragment analysis 7 11 color guide DNA sequencing 2 21 fragment analysis 2 22 Comments column DNA sequencing 6 13 fragment analysis 7 11 Communication P
83. ation see the ABI PRISM GeneMapper Software User Guide Other Software Software Provided with Your Computer If other software programs have been provided on the hard disk of your computer send in the registration cards to receive future updates from their manufacturers The operating system is described in manuals provided with your computer Software Not Provided with Your Computer Do not install other programs on the computer yourself unless directed to do so by a Applied Biosystems representative Other programs may have features that interfere with correct operation of the 310 Genetic Analyzer software 2 16 Overview of the 310 Genetic Analyzer Section Theory of Operation In This Section This section covers the following topics Topic See Page What the 310 Genetic Analyzer Does 2 18 Principles of Operation 2 19 Overview of the 310 Genetic Analyzer 2 17 What the 310 Genetic Analyzer Does DNA Sequencing Fragment Analysis DNA sequencing experiments determine the order of the bases in a DNA sample Fluorescently labeled dyes are attached to ACGT extension products in DNA sequencing reactions Dye labels are incorporated using either 5 dye labeled primers or 3 dye labeled dideoxynucleotide terminators Polymerases such as AmpliTaq 9 FS are used for primer extension The sequencing reaction sample tubes are placed in a tray in the instrument s autosampler The autosampler brings each sample s
84. atrix standards about 6 25 Dye Indicators preferences 4 27 dye set colors and virtual filter fragment analysis 2 22 dye set colors and virtual filters sequencing 2 21 Dye Set Primer column 6 12 dye set primer files installing 4 18 dye set primer files installing 4 18 dye sets and virtual filters 2 20 and wavelengths colors represented by 4 10 choosing for sequencing 6 19 dye terminators excess 6 41 dyes compatibility under virtual filters 2 20 E electrical troubleshooting 8 6 to 8 7 electrodes positioning the capillary near the electrode 3 18 preparing the Cathode electrode 3 23 to 3 26 cleaning 3 23 installing 3 24 straightening 3 26 trimming 3 25 electrokinetic injection 2 19 voltage and time parameters DNA sequencing 6 17 fragment analysis 7 17 electrophoresis 2 19 Electrophoresis History window 4 13 pre injection prerun 4 15 running voltage DNA sequencing 6 18 fragment analysis 7 17 temperature DNA sequencing 6 18 fragment analysis 7 17 electrophoresis buffer See buffer e mail address for technical support 1 9 error messages record of Log file 4 11 to 4 12 error leak detect 3 11 excess dye terminators 6 41 experiments DNA sequencing analyzing data 6 22 cycle sequencing using terminators 6 5 injection list preparing 6 15 to 6 17 materials required 6 7 matrix file creating 6 30 to 6 32 matrix file creating from a sample 6 30 matrix standards preparing 6 25 samples denaturing and loading 6 9 to
85. aults Insufficient free RAM Restart computer before collecting data Note Always restart the computer before collecting data Troubleshooting 8 5 Current Problems 8 6 Troubleshooting Problems with Current Observation Possible Causes Recommended Actions No current Too little or no buffer in anode buffer reservoir Replenish buffer reservoir Too little or no buffer in autosampler position 1 Replenish buffer in position 1 of autosampler Electrode bent a Remove electrode Straighten electrode Replace electrode Recalibrate autosampler Capillary bent away from electrode poops Tape capillary securely to heat plate to keep capillary from shifting position b Recalibrate autosampler Unfilled capillary bubbles in capillary and block a Examine the instrument for leaks See Checking for Polymer Leaks on page 3 11 b Rerun module Pump block is plugged with urea or crystallized buffer Remove and clean block Loose valve fittings or syringe Tighten valve fittings and syringe Anode buffer valve does not open Open buffer valve a Push down on the valve with your finger b Release the valve It should spring to the open position Note Ifthe valve is stuck it should be cleaned See Correcting Leaks at the Valve to the Buffer Reservoir on page 3 12 Plugged broken or nonconducting cap
86. avelengths of the four dyes in each dye set Each of the chemistries is associated with a dye set The relative wavelengths of the dye labels are not consistent across dye sets Therefore the color associated with a base in the raw data depends on the chemistry used to label it The analysis software may reassign the colors to standardize data collected with different labeling chemistries The analysis program converts the information collected by the Data Collection software so that after analysis the color assigned to a base is consistent regardless of the sequencing chemistry used for labelling Color of Analyzed Data Base Blue C Green A Black G Red T Using the 310 Data Collection Software 4 9 Virtual Filters for n DNA sequencing applications the dyes corresponding to the colors in the raw data DNA Sequencing are listed below Virtual Filter A Virtual Filter E Dye Dye dRhodamine Color Primer Terminator Terminators BigDye v 3 0 Blue 5 FAM R110 dR110 Dye 1 Green JOE R6G dR6G Dye 2 Yellow TAMRA TAMRA dTAMRA Dye 3 Red ROX ROX dROX Dye 4 Virtual Filters for In fragment analysis applications the dyes corresponding to the colors are listed Fragment Analysis below Corresponding Dye Dyeset E5 Dye set G5 Dye set D DyesetC Dye set F Color DS 02 DS 33 DS 30 DS 31 DS 34 DS 32 Blue dR110 6 FAM 6 FAM 6 FAM
87. by the DNA sequencing applications and the fragment analysis applications as well as shared consumables Part numbers for many consumables are noted in this appendix Refer to these part numbers when ordering from Applied Biosystems More information about Applied Biosystems kits and consumables is available from your sales representative or on the Applied Biosystems Web site at http www appliedbiosystems com Consumables B 1 Shared Consumables Capillaries Capillaries can be used for up to a minimum of 100 runs The following table lists the capillary types labels and part numbers Length to Internal Capillary Length Detector Coated Diameter Part Type Label Polymer cm cm Uncoated p Number 47 cm x green POP 4 47 36 internally 50 402839 50 um i d POP 6 uncoated GeneScan Polymer 61 cm x pink POP 6 61 50 internally 50 402840 50 um i d uncoated Trays These are the sample trays and accessories used with the 310 Genetic Analyzer B 2 Consumables sample tubes consumables The capillary and electrode can pass through a slit in the septum Tray Consumable Purpose Part Number 96 well MicroAmp9 Holds 0 2 mL tubes for the 96 well tray 403081 tray retainer set 0 2 mL MicroAmp9 Holds samples on the 96 well tray N801 0580 reaction tubes MicroAmp base Holds 0 2 mL tubes for the 96 well tray N801 0531 Adaptor Allows the 9700 The
88. ccording to the values in the module You can edit any parameter by clicking in the field and typing the new value Click another field or press Enter to save the change The default setting for the injection list assumes that each sample will be injected in the order listed in the sample sheet If you prefer a different injection order modify the Tube amp Sample Name column Remember that the samples in the tray must match the positions in the injection list To change the order of samples during a run Step Action 1 You can change the order of injections anywhere in the injection list following the currently running injection The original order of injections RE M x Sample Sheet Sample Sheet042301 0ss v D gt Skip Pause E Cancel Length to Detector fo cm Operator Tube amp Sample Name Test CCD 4 d 0 0 0 Test CCD 4 Color md4 GS STR POP4 1 mL F md4 15 0 Bogus Matrix GS STR POP4 1 mL F md4 15 0 4 Bogus Matrix GS STR POP4 1 mL F md4 15 0 i Bogus Matrix GS STR POP4 1 mL F md4 15 0 Bogus Matrix A11 six GS STR POP4 1 mL F md4 Bogus Matrix ap 2 Select an injection name in the Tube amp Sample Name column and use the drop down list to select the sample you want injected next RE M x Sample Sheet SampleSheet042301 9ss v D gt Skip Pause Bi Cancel Length to Detector fo cm Operator 0 0 0
89. cense or right to perform the above processes This instrument is also licensed under U S patents 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 and the ABI PRISM design AMPFLSTR Applied Biosystems GeneMapper GeneScan Genotyper MicroAmp PROCISE QuantiBlot SeqScape Sequence Navigator and StockMarks are registered trademarks of Applied Biosystems or its subsidiaries in the U S and certain other countries ABI AFPL BigDye CATALYST FAM GeneAssist HEX Hi Di JOE LIZ NED PET POP 4 POP 6 ROX TAMRA TET and VIC are trademarks of Applied Biosystems or its subsidiaries in the U S and certain other countries AmpliTaq AmpliTaq Gold GeneAmp and TaqMan are registered trademarks of Roche Molecular Systems Inc All other trademarks are the sole property of their respective owners Applied Biosystems is committed to providing the world s leading technology and information for life scientists Printed in the USA 06 2010 Part Number 4317588 Rev B Contents 1 About This Guide Introduction oro net ebesveRbiesht gebe bee ginko cre ERE teh tied baa ed eee 1 1 In This Chapt
90. ch Technical Support Through the Applied Biosystems Web Site 1 12 To Obtain Technical Documents 0 0 0 1 12 To Obtain Customer Training Information 00 00 e eee eee eee 1 13 2 Overview of the 310 Genetic Analyzer IrtroductlOfi zoo if bathe tick ee habe AM ga ee ee In RES eed Sabb uate 2 1 In This Chapter esine sees fee sages Cad diiven 1833 GSERUSG be TEA ae Gers 2 1 About This Chaptet sz beu st shi bei RET pa eae ea ba hase 2 2 For More Information ere tuk eee ES SS oe eee Ee DE 2 2 Section Description of the Instrument 1 0 1 0 0 00 ccc cee enhn 2 3 In This Section c4 6 e500 cnn ee bhai ayy ie ee ae bee a 2 3 Front 6f the Instruments 03 scars Baas ES npe OR ER RR ee ie ba RC dE UE 2 4 Diagram s n jesus aria Seti aS ages TL 2 4 Status INdiCators s 2 2 cree ae etek ee bo eet REND EER RA ESEEEYPILEES 2 4 Back of the Instrument verser eee acne gate x RR ale EEEE ede 2 5 Didgr i ic pe BU ESACUNL Me PE DEP PEE HET RE PENES 2 5 Service BEDS cus sac p S RR pA ER CR GENDER ROBUR PS als 2 5 Serial Connections 2 2 06 ce cece eee ehh he hm hh hh 9 e hen 2 6 Reset Button eur eneee ewe oe ree Tee Gta eh eee One tab eaten de PETRUS 2 6 On Off S Witch cso ERU D EE Satins SUR ERR RU AEn RR cree Biel Ack 2 6 Power Cord Receptacle 2 i Gea eee bI Deor Ren v vU US ERES 2 6 Behind the Doors of the Instrument 20 0 0 cece eee e 2 7 OVerVIGW v eod aan Ahaha he RCM a a em ren
91. ctrophoresis injects the sample into the capillary The autosampler then moves the cathode buffer reservoir to the capillary and electrode to continue electrophoresis The cathode buffer reservoir a waste vial and a water reservoir used to wash the cathode electrode and capillary tip between samples are vials located on the autosampler The short period of electrophoresis conducted while the capillary and cathode are immersed in the sample is called electrokinetic injection A portion of the sample forms a tight band in the capillary during this injection Electrophoresis is the movement of charged molecules through a polymer in an electrical field It is used to separate DNA fragments by size Samples are electrophoretically separated as they travel through polymer in the capillary FWULNXUM HIGH VOLTAGE HAZARD Exposure to high voltage can cause serious injury and death Do not touch the electrodes or capillary when the electrophoresis power supply is on An interlock switch shuts off the electrophoresis power supply if the front doors of the instrument are open Since the capillary is fastened along most of its length to a heat plate temperature is controlled during electrophoresis As the DNA fragments pass through the window of the capillary an argon ion laser excites the attached dye labels and they fluoresce NUNN LASER HAZARD Exposure to direct or reflected laser light at 10 mW for 0 1 seconds can burn the retina and l
92. cturer and that ramping rates are 1 C second BigDye Terminators The procedures given here are for the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits Refer to the Automated DNA Sequencing Chemistry Guide for information about other chemistries 6 38 Setting Up DNA Sequencing Experiments Cycle Sequencing 1X Reactions Reaction Types Step Action 1 For each reaction add the following reagents to a separate tube Reagent Quantity BigDye Terminator Ready Reaction Mix 8 0 uL Template 7 single stranded DNA 50 to 100 ng double stranded DNA 200 to 500 ng PCR product DNA 2 to 100 ng depending on size Primer 3 2 pmol Deionized water q s Total Volume 20 uL Mix well and spin briefly If using the DNA Thermal Cycler TC1 or DNA Thermal Cycler 480 Overlay the reaction mixture with 40 uL of light mineral oil Setting Up DNA Sequencing Experiments 6 39 About Cycle Sequencing Overview Conditions for BigDye Terminators Cycle Sequencing on the GeneAmp Thermal Cyclers These protocols have been optimized for all Applied Biosystems thermal cyclers including the DNA Thermal Cycler TC1 the DNA Thermal Cycler 480 the CATALYST 800 Molecular Biology LabStation the 877 Integrated Thermal Cycler and the GeneAmp PCR Systems 9700 9600 and 2400 The protocols contained in this section should work for all seven instruments If you use a
93. d raw data can be used to create a matrix file Note BigDye v 3 matrix standards differ from previous versions of matrix standards If you are using BigDye v 3 matrix standards follow the protocol outlined in the product insert to generate a matrix for use with BigDye v 3 chemistries The following are the matrix standards used for DNA sequencing ABI PRISM dRhodamine Matrix Standards Kit dR6G dR110 dTAMRA dROX LIZ 310 377 v 3 0 BigDye Matrix Standards Kit Note For part number information see Appendix B Consumables For this example the dRhodamine matrix standards are used for generating a matrix for most chemistries run with Virtual Filter Set E BigDye terminator v 1 0 and v 2 0 BigDye Primer v 1 0 dRhodamine terminator The dRhodamine matrix standards are Tube Label Color of Raw Data Base dR110 Matrix Standard black G dR6G Matrix Standard green A dTAMRA Matrix Standard blue C dROX Matrix Standard red T Matrix standards are stable for 6 months at 2 to 6 C Avoid freeze thaw cycles The dRhodamine matrix standards are provided in a ready to use format and are premixed with a blue dye for use on slab gels The blue dye is not necessary nor is it detrimental to use it with the 310 Genetic Analyzer The colors in the generated matrix analyzed data are consistent with current base calling conventions i e C is blue A is green G is black and T is red in ana
94. dye sets 2 20 for DNA sequencing and fragment analysis 4 10 virtual filters colors and dyes DNA sequencing 2 21 fragment analysis 2 22 voltage and time parameters DNA sequencing 6 17 fragment analysis 7 17 voltage spikes how to avoid 3 42 W water replacing when to 3 10 wavelengths represented by virtual filters 4 10 Y yield gel using to evaluate sample 8 20 Index 7 Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For sales office locations and technical support please call our local office or refer to our web site at www appliedbiosystems com Applied KS Bibsystems Applied Biosystems is committed to providing the world s leading technology and information for life scientists 06 2010 Part Number 4317588 Rev B
95. dyes There will be either four or five colors depending on your dye set Set the colors in the Dye Indicators preferences Std A marker appears in this column to indicate that a particular dye in your sample is a standard The field should be empty when running matrix standards and filled under most other circumstances If you do not choose to launch and run the GeneScan Analysis software automatically or if you reanalyze data later you can make changes to this selection through the analysis software Raw data is collected for all colors but only the colors for which a sample is present are automatically analyzed by the GeneScan Analysis software Setting Up Fragment Analysis Experiments 7 11 Using a Previously f you are running the same group of samples for a second or third time you can use Created Sample the original sample sheet Select the previously created sample sheet when filling out Sheet the injection list To modify an existing sample sheet Step Action 1 Open the Sample Sheet It is stored in the Sample Sheet folder 2 Make changes and save the Sample Sheet To keep the original choose Save As and give the sample sheet a new name To overwrite the original choose Save without renaming the Sample Sheet 3 Select the modified Sample Sheet when filling out the Injection List Importing Sample You can import data from tab delimited text files into the grids of the sample
96. e To pause or cancel a running module click the Pause or Cancel buttons Using the 310 Data Collection Software 4 7 Raw Data Display Overview During arun you can monitor Real time raw data Instrument status Instrument errors Real time electrophoresis data Raw Data Window The Raw Data window shows data detected as it passes the capillary window with a different colored line representing each filter The computer simultaneously updates the Raw Data window with four lines every few seconds during instrument operation To open the Raw Data window select Raw Data from the Window menu ERN Data one o x 9 Zeon aron e 2 8 192 7 168 6 144 5 120 4 096 3072 2 048 The raw data window will display either four or five lines of data depending on the number of dyes being run Resizing Raw Data Double click the left side Y axis of the Raw Data window to display the Set Scale dialog box where you can enter values to scale the raw data Set Scale xi Maximum value o216 0 Minimum value foo Cancel OK To change the scale for Scans Time double click the top X axis of the Raw Data window to display the dialog box 4 8 Using the 310 Data Collection Software Color Buttons Why Raw Data and Analyzed Data Differ in Color Data Display after Analysis for DNA Sequencing The buttons at the bottom of the Raw Data w
97. e injection list The syringe encoder indicates that the syringe is located more than 500 steps from the home position a Remove syringe and pump block b Clean the pump block c Clean the syringe Injection is cancelled an alert appears on the computer screen Instrument ran out of polymer during the injection Check the log file Injections that last 1 to 2 minutes and out of polymer errors are recorded there when the syringe empties Buffer reservoir is filling up Leaks at the valve to the buffer reservoir Check for leaks at the valve to the buffer reservoir See Correcting Leaks at the Valve to the Buffer Reservoir on page 3 12 Bubbles in the glass syringe Remove bubbles from syringe Automatic Data Analysis Problems Problems with Automatic Data Analysis Observation Possible Causes Recommended Actions Data was not automatically analyzed Sample sheet not completed or completed incorrectly Review and correct sample sheet Injection list not completed or completed incorrectly Complete injection lists as described Analysis preferences set incorrectly in the ABI PRISM 310 Data Collection Software Check the Data Collection software preferences Select Autoanalyze with the AnalyzeGSSample bat under the GeneScan Injection List Defaults Select Autoanalyze with AnalyzeSampleFile bat under the Sequencing Injection List Def
98. e barrel as a matched set to Storing Syringes ensure that the syringes Recommendations Do not leak Have the proper breakaway force Note Breakaway force is the pounds of normal force not psi needed to start the plunger in motion Interchanging plungers and barrels can result in Leakage Shortened syringe life breakaway force too high or too low Capillaries not filled completely breakaway force too high Keep these important facts in mind Do not place O rings on the syringe plunger Doing so will alter the syringe breakaway force and cause improper filling of capillaries Do not rapidly heat or cool an assembled syringe as doing so will crack the glass barrel Allow the polymer to reach room temperature before filling the syringe Remove the plunger from the barrel slowly count slowly to 5 Moving the dry plunger quickly in the barrel may damage the Teflon plunger and cause leakage around the plunger Resting the plunger on the lab bench may change the shape of the Teflon tip Do not store the syringe barrel and plunger separately Store syringes Dry Away from direct sunlight With the plunger in the barrel Do not lubricate the plunger with polymer before inserting it into the barrel Lubricating with polymer can damage the Teflon plunger Use only deionized water to lubricate the syringe Operating the Instrument Hardware 3 39 3 40 Operating the Instrument Hardware
99. e capillary when it will not be reused within 1 week Step Action 1 Flush the capillary free of polymer as follows Rinse the gel block Fill the glass syringe with 200 uL of water Close all valves Move the syringe drive to position 250 Open the Data Collection software if not open already From the Manual Control window run the Seq Fill Capillary module to force the polymer out of the capillary 0000 9 t takes about 6 minutes to force the polymer from the capillary Note When the capillary is completely flushed of polymer the water flowing through the capillary may trigger the leak detector If this happens click OK in the Pause Alert window resume the run from the menu and cancel the run immediately Remove the capillary by following the installation steps in reverse order Clean the gel block After Reinstalling a Stored Capillary After reinstalling a stored capillary be sure you Runaknown standard to verify the condition of the capillary Recalibrate the autosampler Operating the Instrument Hardware 3 21 Verifying that the Capillaries used with Performance Optimized Polymers are uncoated A proper coat of Capillary Fills polymer is especially important for these capillaries Routinely check that capillaries fill Correctly Properly To determine if the capillary is filling properly check the Log File The syringe drive encoder should move one
100. e computer collects the data interprets it and stores it on the computer s hard drive Data Collection The File Edit Instrument Window and Help menus are available when you open the Software Menus Data Collection software FE 310 Data Collection Software Version 3 0 0b1c11 File Edit Instrument Window Help Data Collection The following table lists the functions you can do with the Data Collection software Functions Monitor Instrument Status Raw data Electrophoresis History Instrument Log Automatically Start the data analysis software Direct analysis programs to print the data Manage files by Editing modules Importing and exporting data Four Key Tools in To run the 310 Genetic Analyzer successfully you need to know how to operate four the Software key tools in the Data Collection software Manual Control Raw data display Sample sheet Injection list Manual control and raw data display are discussed later in this chapter See Chapter 6 Setting Up DNA Sequencing Experiments and Chapter 7 Setting Up Fragment Analysis Experiments for information about sample sheets and injection lists See Chapter 5 Injection Lists for additional information about injection lists 4 4 Using the 310 Data Collection Software File Types The following table lists the types of software files that are required by the instrument Application Softwa
101. e damage to the central nervous system and the male and female reproductive systems and is a possible birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To prepare matrix standard samples Step Action 1 For each sample mix in a sample vial a 1 0 uL matrix standard b 12 0 pL deionized formamide Label each vial according to the dye Gently vortex the mixture for 3 to 5 seconds Spin down the mixture o AeA OJN Store at 2 to 6 C until ready to use Setting Up Fragment Analysis Experiments 7 23 Denaturing the You can denature the matrix standard samples in the thermal cycler with your samples Matrix Standard or follow this procedure Samples Step Action 1 If you are using the GeneAmp PCR System 9600 or 9700 to denature your samples plan the order in which you will place the samples in the 96 well tray Note Each color must have a separate well in plate Do not mix matrix standards into one well Here is a suggested configuration S Matrix standard samples are in A1 through A4 tr QO W ri T Q e O O O O O O OOOOOOOS OVVVO OOOOOOOS QUOCOCOCOD OOOOOOQCQs QUOODOCCGCLCS OJOJ 2900p 616 6 OOOOOOQOO OOOOOOQOO OOOO 2 Heat the sample for 5 minutes at 95 C 3 Chill the sample on ice 4 Hold on ice until ready to load in
102. e instrument 4 Hold the Tray button down for 10 seconds while you turn on the instrument This deletes the firmware The three lit LED status lights indicate the absence of firmware 5 Restart the computer and open the Data Collection software A dialog box appears as the firmware reloads Open the Manual Control window select Syringe Home and click Execute In the Manual Control window select Autosampler Home X Y and click Execute Autosampler Home Z and click Execute Operating the Instrument Hardware 3 47 Performing a Clear IMPORTANT Since a clear memory reset will delete the calibration data it is imperative that Memory Reset YOU record the current calibrated values If you forget to record this data before a clear memory reset a service engineer will need to reestablish the calibration values You can find calibration data on the Calibration sticker through Manual Control or through LabView software In this procedure you will record the values manually and as a calibration file To record the calibration data Step Action 1 Open Manual Control and record the values for CCD Pixel Position X CCD Pixel Position Y Syringe Pump Force Syringe Max Travel In Manual Control select Calibration File Make Quit the Data Collection software and shut down the computer Turn off the instrument To perform a clear memory reset Step Action
103. e sample name A drop down list appears 4 GeneScan Injection Sheet Untitled 2 x Sample Sheet Sample Sheet GSscreenshots D D Run Pause Wi Cancel Length to Detector fo cm Operator Inj Tube amp Sample Hame Module Inj Secs ini v Run kV Run C Run Time Matrix File A1 Sample 1 GS STR POP4 1 mL F md4 5 150 150 24 Bogus Matrix aj 1 2 A3 Sample 2 GS STR POP4 1 mL F md4 5 15 0 15 0 E 24 Bogus Matrix 3 AS Sample 3 z es STR POP4 1 mL F md4 5 150 15 0 24 Bogus Matrix 4 Bogus Matrix l gt b 3 Select the sample you want to inject In this example instead of injecting a sample in position A5 of the autosampler as the third injection sample in position A3 will be the third injection Ed GeneScan Injection Sheet Untitled 2 _ x Sample Sheet Sample Sheet GSscreenshots D p Run Pause Wi Cancel Length to Detector fo cm Operator inj Tube amp Sample Name Module Inj Secs ini kV Run kV Run C Run Time Matrix File 1 A1 Sample 1 GS STR POPA4 1 mL F md4 15 0 15 0 24 Bogus Matrix aj 2 A3 Sample2 GS STR POP4 1 mL F md4 15 0 15 0 24 Bogus Matrix 3 Jas Sample 3 z GS STR POP4 1 mL F md4 15 0 15 0 24 Bogus Matrix 4 Bogus Matrix l gt L4 Adding Rows toan The injection list has the same number of rows as the sa
104. e the autosampler Note Always recalibrate the autosampler if the cathode It is placed in the sample during electrophoresis electrode is removed You can remove and replace the cathode electrode replaced or cleaned Cleaning the When to Clean the Electrode Electrode Glean the electrode every 48 hours In addition you will need to clean the electrode in any of the following situations Ifit has been touched Ifitis new When autosampler buffer solutions are replaced Note Crystals formed on the electrode can fall into the sample and clog the capillary Since the capillary is removed from buffer while the electrode is cleaned complete the procedure quickly so that the capillary does not dry out How to Clean the Electrode To clean the electrode Step Action 1 Open the Data Collection software if not open already 2 Press the Tray button on the 310 Genetic Analyzer to lower the autosampler and present the tray 3 Wipe the electrode with lint free lab wipe that has been dampened with distilled deionized water Dry the electrode with fresh lint free lab wipe 5 Press the Tray button to return the autosampler to its original position and immerse the capillary in buffer 6 Recalibrate the autosampler after cleaning trimming or replacing the electrode Operating the Instrument Hardware 3 23 Installing the To install the electrode Electrode Step Actio
105. e to fill the capillary with polymer Luer valve Attaches the plastic syringe to the pump block Capillary ferrule Attaches the capillary to the gel block Waste valve Allows for waste flow when priming and cleaning the gel block channels Waste vial optional Collects waste put through the waste valve Overview of the 310 Genetic Analyzer 2 9 Detection Region The detection region collects data from the samples as they migrate past the capillary window Parts of the detection region Capillary Heat plate Thermal tape Detector window Detector door GR0982 Part Purpose Capillary Carries the sample past the laser to the gel block Heat plate Heats the capillary during electrophoresis Heater door Insulates the capillary during electrophoresis and protects you from burns Thermal tape Holds the capillary in place against the heat plate Detector window Allows the laser to pass through the capillary window and excite the dye labels attached to the sample Detector door Holds the capillary in place and protects you from exposure to the laser a The heater door covers the heat plate It is not shown in the drawing above 2 10 Overview of the 310 Genetic Analyzer Autosampler Region The autosampler moves buffers and samples to the capillary Electrode thumbscrew Cathode electrode 48 or 96 well tray
106. eave a permanent blind spot Never look directly into the laser beam or allow a reflection of the beam to enter your eyes The laser is located behind the detector door and the front door of the instrument An interlock switch protects you from the laser when the front door of the instrument is open Overview of the 310 Genetic Analyzer 2 19 Detection A series of lenses direct and focus the fluorescent light into a spectrograph A diffraction grating in the spectrograph disperses the light by wavelength and focuses the resulting light spectrum onto a CCD array Data Collection The Data Collection software collects raw data and allows real time run monitoring For more information about data collection see Chapter 4 Using the 310 Data Collection Software Virtual Filters and The Data Collection software defines certain areas on the CCD array for the collection Dye Sets of the fluorescent emissions from the dye labels in the dye set These areas are called virtual filters There can be any number of virtual filters since the filter is simply a software designated site on the CCD array Virtual filters are grouped into sets and referred to by a letter such as Virtual Filter Set E5 G5 E D C or F Fluorescent dye labels come in sets of four or five There are several different dye sets for use in different types of experiments The fluorescence from each dye set must be collected using the correct virtual filter set You select t
107. ecommended Not Germline Mutations 50 50 Recommended Comparative Sequencing Recommended Not Recommended Recommended Not Somatic Mutations 30 70 Recommended Recommended DNA Sequence Context G C rich gt 65 Recommended Satisfactory Recommended Recommended Satisfactory A T rich gt 65 Recommended Recommended Recommended Recommended Satisfactory G T rich regions Not Satisfactory Recommended Recommended Satisfactory Recommended Homopolymer A or T gt 25 bp Not Recommended Recommended Recommended Satisfactory Recommended GA Motifs Recommended Satisfactory Recommended Recommended Satisfactory Template Plasmid 15 bp Recommended Recommended Recommended Recommended Satisfactory M13 Recommended Recommended Recommended Recommended Satisfactory BAC Cosmid Lambda XL Recommended Satisfactory Satisfactory Not Not PCR Recommended Recommended Bacterial genomic DNA Recommended Not Not Not Not Recommended Recommended Recommended Recommended PCR Amplicon Recommended Recommended Recommended Recommended Satisfactory PCR Amplicon Recommended Satisfactory Recommended Recommended Not Heterozyous 50 50 Recommended PCR Amplicon Recommended Not Recommended Recommended Not Heterozyous 30 70 Recommended Recommended 6 6 Setting Up DNA Sequencing Experiments Performing a DNA Sequencing Experiment Materials Required To run a fragment analysis experiment you will need t
108. ects the instrument to the COM1 port on the computer DIAGNOSTICS Connects the instrument to a diagnostics device or the computer communications port AUX SERIAL Auxiliary serial connection This button resets communications between the instrument and the computer Insert a pen or a similar thin object to push the recessed button and activate the reset For more information see Resetting the Genetic Analyzer on page 3 46 This switch turns power to the instrument on and off A power cord delivers power to the instrument from a wall receptacle and is rated for 110 or 230 VAC For information about the power see the ABI PRISM 310 Genetic Analyzer Site Preparation and Safety Guide 2 6 Overview of the 310 Genetic Analyzer Behind the Doors of the Instrument Overview Hardware located inside the 310 Genetic Analyzer is responsible for Automated sample handling Electrophoresis Fluorescence detection Some of this hardware is located directly behind the doors Other parts such as power supplies electronics detection optics and the laser are only accessible by a service technician Diagram Gel block region Detection region Autosampler region Overview of the 310 Genetic Analyzer 2 7 Gel Block Region The gel block controls the flow of polymer and samples through the capillary Syringe drive Syringe drive toggle UTION CA
109. ee Mg Total Mg2 Total dNTP Incorrect pH Verify buffer pH and buffer concentration Wrong PCR tube Use GeneAmp Thin Walled Reaction Tubes for the DNA Thermal Cycler 480 MicroAmp Reaction Tubes with Caps for the GeneAmp PCR System 9700 9600 and 2400 MicroAmp Base used with tray retainer set and tubes in GeneAmp PCR System 9700 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 DNA Thermal Cycler 480 a Push reaction tubes firmly into contact with block after first cycle b Repeat amplification GeneAmp PCR System 9600 heated cover misaligned Align the 9600 heated cover so that white stripes align after twisting the top portion clockwise Troubleshooting 8 19 8 20 Troubleshooting Problems with Poor Amplification continued Observation Possible Cause 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 amp
110. ee e eee en 2 7 Dia stain coe iiss ee RI wich kha be NR RUN ES E NE E beer ERES 2 7 Gel Block Region ide pe Eo Rp Cr b DRURU I Ra Rea RR 2 8 Detection Region ooco e a PAL seat RV PPM CLR IX URS 2 10 Autosampler Region Anis ki vines Leer RS Gia ie see bs aes EP ERIS 2 11 Section Description of the Computer and Software eee 2 13 In This Section 42 t o RR Re He nee Serene aa Ehe vU 2 13 Computer sits ent o ite bt sale EGRUS DIN EI EDEN EISERONES IS 2 14 Oye rvi EW ie ee ARRIERE Wa aa Ep vetet eie ex reden EAT EAE e ERE ea 2 14 System Requirements eierens osni oaa eee e 2 14 RAM Requirement 5 raa se ERR ERREUR UN UR Ran ORAS Date CURL ASEA 2 14 OMWALE c ree Pa em Pe DI RR EUR 2 15 OVERVIEWS cues e ERES REUS GERI ESAE UE ED bi Se ns Rr o e 2 15 Software for Data Collection 0 0 cece eee ne 2 15 Software for Data Analysis 1 0 0 0 ec ee 2 16 Other Software css uvseenp certetenfeebnvzver e RUE ade tre ES 2 16 Section Theory of Operation ccce eer hn 2 17 In This Section se penei n a sob dig So db Meg tegeret t 2 17 What the 310 Genetic Analyzer Does 00 0 0 0 cee eens 2 18 DNA Sequencing ee pM SEP UEM P EAE LqUMP CR 2 18 Fragment Analysis coepere b eEReeUL eer eerie ew bre a ee rU 2 18 Principles of Operation sas eh SE be ed a IE OR RR BSA ts ER 2 19 sample Labeling avast at ae aaa I rex gerere 2 19 Automated Sample Handling 0 0c eee ene 2 19 Electrokinetic Sample Inject
111. efficiency by Adjusting screen variables such as brightness contrast and color to suit personal preferences and ambient lighting Positioning the screen to minimize reflections from ambient light sources Positioning the screen at a distance that takes into account user variables such as nearsightedness farsightedness astigmatism and the effects of corrective lenses When considering the user s distance from the screen the following are useful guidelines The distance from the user s eyes to the viewing screen should be approximately the same as the distance from the user s eyes to the keyboard For most people the reading distance that is the most comfortable is approximately 20 inches The workstation surface should have a minimum depth of 36 inches to accommodate distance adjustment Adjust the screen angle to minimize reflection and glare and avoid highly reflective surfaces for the workstation Use a well designed copy holder adjustable horizontally and vertically that allows referenced hard copy material to be placed at the same viewing distance as the screen and keyboard Keep wires and cables out of the way of users and passersby Choose a workstation that has a surface large enough for other tasks and that provides sufficient legroom for adequate movement Technical Support Contacting Technical You can contact Applied Biosystems for technical support Support By e mail
112. er esceresesbkete M bs eet et RUNS DERE DHS SEER Ob SEES 1 1 About Ehis Guide coe phECPDERME ooh cited E WC RE Goat aos eb ee 1 2 Overview of Contents s cics cece cia ote ee Galas ER Y VR REC ahs 1 2 About the Quick Reference Card 0 0 0 0 ccc cece eens 1 3 Related Documents kb eee ih A a i eee ag 1 3 Safety ess cse cLCUAMDepBRESRUSOPOUNi ed Bates Gia bie ERG ME T DEVePEPRUDS PEDE 1 4 Documentation User Attention Words 0 0 cece eee ee eee 1 4 Chemical Hazard Waming 0 0 ec eect eee 1 4 Chemical Waste Hazard Warning 0 0 cece eee eee nee 1 5 Site Preparation and Safety Guide 0 0 eee eee 1 5 About MSDS So nrt e teena hte ik od dices tbe e didnot deeded eh hate ts 1 5 Ordering MSDSS cis Se ERA ae EC deed qum aha eee eats ER 1 6 Instrument Safety Labels 02 0 0 E E nee eee nee 1 6 About Waste Prohlles v si snie RR eee v SW PY we Ga eer eek E nO eee des 1 7 About Waste Disposal sesasi peniteat nepra eee nent hme 1 7 Before Operating the Instrument seeleeeeeeeeee e 1 7 Safe and Efficient Computer Use 1 7 Technical Support obo ovo bu EP RUEDAS EIAS 1 9 Contacting Technical Support 0 0 ee eee ee eee 1 9 To Contact Technical Support by E Mail 0 00 0 0 eee eee eee 1 9 To Contact Technical Support by Telephone or Fax North America 1 9 To Contact Technical Support by Telephone or Fax Outside North America 1 11 To Rea
113. er File 6 19 Starting the Run 6 22 Setting Up DNA Sequencing Experiments 6 3 About Sequencing Analysis PCR and Although the reaction mixes and temperature cycling are similar it is important to Cycle Sequencing understand the difference between PCR and cycle sequencing PCR uses two primers and a double stranded template to amplify exponentially a sequence of interest The template is denatured the primers are allowed to anneal to the template and the primers are extended The purpose is to amplify the unknown sequence for further experimentation such as sequencing Cycle sequencing can use one or two primers and a single stranded or double stranded template The purpose is to determine the base sequence of a DNA template In cycle sequencing the temperature cycles from a denaturation temperature of about 95 C to an annealing temperature of about 56 C The primers attach to complimentary regions of the denatured template The temperature is raised to 72 C and Taq polymerase extends the primer incorporating ddNTPs that stop the extension reactions This process generates fragments randomly that differ in length by one base This temperature cycling can continue without the addition of enzyme because AmpliTaq9 DNA Polymerase FS tolerates high temperatures In primer sequencing the primers are fluorescently labeled in terminator sequencing the ddNTPs are fluorescently labeled 6 4 Setting Up DNA Sequencing Experimen
114. er the tube with a septum and vortex well oc A O N Heat the TSR mixture for 2 minutes at 95 C and place it on ice until ready to place in the instrument Loading the Samples To move the samples into the autosampler Step Action 1 If necessary transfer the denatured samples to a 48 or 96 well tray IMPORTANT The tube arrangement and order of the samples in the tray and on the sample sheet must be the same Make note of the tube arrangement you use so that you can prepare the sample sheet correctly Seal each tube with a septum and place the tray into the autosampler For more information about preparing the sample trays see Preparing the 96 Well Sample Tray on page 3 33 6 10 Setting Up DNA Sequencing Experiments Preparing the DNA Sequencing Sample Sheet About Sample Sheets Preparing the Sample Sheet The sample sheet associates sample information name and type of analysis with a sample tube position in the autosampler Fill out the sample sheet in the Data Collection software to name each sample associate it with a position in the autosampler and make some choices about the sample s processing There are two kinds of sample sheets for DNA sequencing Sequencing 48 sample tubes Sequencing 96 sample tubes The first time you use any group of samples you must create a sample sheet The sample sheet will be saved in the Sample Sheet folder on the compu
115. erences for automatic file naming can be entered in this part of the Data Collection software Preferences TU crasen seeds Injection List date x ow Select none today s date or global serial number as a file name suffix from the drop down lists IMPORTANT The names you enter into the fields can only have alphanumeric characters The software does not allow non alphanumerics such as lt gt V because such characters can disrupt the automatic creation of files Using the 310 Data Collection Software 4 21 Sequencing Sample When you create a new Sequencing sample sheet a portion of the form is Sheet Defaults automatically filled in for you The information comes from a preference file which you Preferences Can modify Preferences For Sequencing sample sheets you can set these sample sheet preferences Preference Description Dye Set Primer Default dye set primer file that will automatically appear on a newly created sample sheet Matrix Default matrix file that will automatically appear on a newly created sample sheet 4 22 Using the 310 Data Collection Software GeneScan Sample When you create a new GeneScan sample sheet a portion of the form is Sheet Defaults automatically filled in for you The information comes from a preference file which you Preferences Can modify Preferences For GeneScan sample sheets yo
116. ext electrophoresis to be sure the capillary is carrying current Polymer may have dried in the capillary and created a blockage that requires cleaning or replacement Operating the Instrument Hardware 3 49 Switching Between DNA Sequencing and Fragment Analysis Switching Between The 310 Genetic Analyzer can be used for both DNA sequencing analysis and Applications fragment analysis Each time you switch between these two applications you must prepare the instrument by doing the following Take this action See page Cleaning the Syringe 3 35 Removing and Storing the Capillary 3 20 Cleaning the Gel Block 3 27 3 50 Operating the Instrument Hardware Maintaining and Caring for Your Computer Overview Hard Disk Maintenance Back Up All Programs and Files Regularly Use a Hard Disk Maintenance Program Regularly Use Discretion When Adding Software Programs Computers require regular attention and maintenance to operate efficiently and consistently Because the software for the 310 Genetic Analyzer on the computer works with large files and accesses the hard disk often it is especially important to follow the procedures described here to minimize errors during operation Follow these guidelines for optimal performance of your 310 Genetic Analyzer software and the computer Back up all programs and files regularly Use a hard disk maintenance program regularly to reorganize a fragmented disk Use d
117. fer to Run Status and History on page 4 11 for information on monitoring Analyzing the Data The Sequencing Analysis software automatically analyzes the samples when the run finishes if the automatic analyze checkbox on the Sample Sheet is checked See the ABI PRISM DNA Sequencing Analysis Software User Guide for more information 6 22 Setting Up DNA Sequencing Experiments Section Matrix Files for DNA Sequencing In This Section This section covers the following topics Topic See Page About Matrix Files 6 24 Preparing Matrix Standards 6 25 Creating Matrix Files from Matrix Standards 6 27 Creating a Matrix from a Sample 6 30 Setting Up DNA Sequencing Experiments 6 23 About Matrix Files Matrix Files How Many Matrix Files About Using Matrix Files Matrix files contain information that corrects for spectral overlap Spectral overlap occurs when part of one dye s emission spectrum falls onto a portion of the detection hardware that is collecting the fluorescent peak of another dye Dye sets are created to minimize spectral overlap but it still occurs to some extent When choosing matrix files in the sample sheet match the matrix file to the samples by dye set and sequencing chemistry For example samples run with a module file for Virtual Filter E should be analyzed with a matrix file that was also run with Virtual Filter E The dye set used to create the matrix file must be the same as the
118. ferences Preference Description Length to Detector The distance between the end of the capillary that is in the sample and the capillary window Enter the length that will automatically appear on a newly created injection list The length you enter does not affect the configuration of the instrument It is for your record keeping purposes only Operator Enter the name of the operator that will automatically appear on a newly created injection list 4 Dye Module A module file contains the instructions that tell the 310 Genetic 5 Dye Module Analyzer how to run a sample It includes such things as injection time voltage and heat plate temperature Select the default module file that will automatically appear on a newly created injection list Matrix A matrix file contains an algorithm that adjusts the data for spectral overlap of the fluorescent peaks in a dye set Select the default matrix file that will automatically appear on a newly created injection list Autoanalyze with Analyzes your samples automatically using the analysis program you specify in the drop down list From the drop down list select the software program e g AnalyzeGSsample bat you want to use to analyze your data Using the 310 Data Collection Software 4 25 For GeneScan injection lists set the following preferences continued Preference Description Analysis Parameters file Holds the default start and s
119. ge 8 3 Setting Up DNA Sequencing Experiments 6 37 About Preparing Sequencing Reactions Reagent Age and The freshest reagents are likely to perform the best The following methods are Reaction Storage recommended for guaranteeing reagent freshness Store reagents at 15 to 25 C when not in use and thaw completely at room temperature or in an ice bath do not heat before use Note Do not use a frost free freezer The automatic cycling of the temperature for defrosting can damage reagents Avoid excess more than ten freeze thaw cycles Aliquot reagents in smaller amounts if necessary Shield reagents and sequencing reactions from light Fluorescent dyes are susceptible to bleaching If you would like to store sequencing reactions for future use purify and dry them Store at 15 to 25 C in a non frost free freezer Reaction Tubes The type of tube required depends on the type of thermal cycler used For the DNA Thermal Cycler TC1 and DNA Thermal Cycler 480 use 0 5 mL GeneAmp Thin Walled PCR tubes For the GeneAmp PCR Systems 9700 9600 and 2400 use 0 2 mL MicroAmp PCR tubes If using the CATALYST 800 or ABI PRISM 877 Integrated Thermal Cycler refer to your instrument user s manual for reaction setup Thermal Cyclers The type and performance of the thermal cycler used to prepare sequencing reactions can affect the quality of the reactions Ensure that the thermal cycler is calibrated regularly by the manufa
120. h of the samples Note The first time you run sequencing reactions under new conditions any matrix file can be used for the run After the run you must make a matrix file for the new conditions Note _ If there is no matrix file selected the sample will not be autoanalyzed 6 12 Setting Up DNA Sequencing Experiments Using a Previously Created Sample Sheet To create a new DNA sequencing sample sheet continued Step Action 8 In the Collection Name column select a collection name for the sample This is necessary only if you will be uploading the samples into the Sequence Collector software Note f you are using collection names you must first enter them in the Collection Info Preferences window see Collection Info Preferences on page 4 28 9 In the Comments column enter any additional comments that you want to link to the samples 10 From the File menu select Save As 11 Name the sample sheet and press Return to save it in the Sample Sheets folder If you are running the same group of samples for a second or third time you can reuse the original sample sheet Select the previously created sample sheet when filling out the injection list To modify an existing sample sheet Step Action 1 Open the sample sheet It is stored in the Sample Sheet folder 2 Make changes and save the sample sheet To keep the original choose Save As and give the sample sheet a new na
121. he tube 8 Inspect the depth of the capillary and electrode in the tube visually Homing the When to Home the Autosampler Autosampler Home the autosampler after Turning on the main power switch Resetting the 310 Genetic Analyzer How to Home the Autosampler To home the autosampler Step Action 1 Open the Data Collection software if not open already 2 From the Window menu select Manual Control 3 6 Operating the Instrument Hardware To home the autosampler continued Step Action 3 From the Function drop down list select Autosampler Home X Y Axis then click Execute IMPORTANT Always home the X Y positions before the Z position Homing the Z position first can bend the electrode Manual Control Xx Function 11 Value Range EXSCUS autosampler Home X Y Axis gt No Value No Range Autosampler Present Tray Autosampler Return Tray Autosampler Home X Y Axis Autosampler Home 2 Axis Autosampler To Position Autosampler Up Autosampler Down Buffer Valve Open Buffer Valve Close Electrophoresis On From the Function drop down list select Autosampler Home Z Axis then click Execute Manual Control Xx Function 14 Value Range Seana autosampler Home Z Axis gt No Value No Range Autosampler Present Tray Autosampler Return Tray Autosampler Home X Y Axis Autosample
122. he following items For part number information for the consumables see Appendix B Consumables Lab Equipment Filter sterilized deionized water Disposable non powdered chemical resistant gloves Microcentrifuge variable speed capable of reaching 14 000 x g Table top centrifuge with 96 tube tray adaptor Vacuum centrifuge Savant Speedvac or equivalent Vortexer Small volume calibrated pipette and tips we recommend the Gilson Pipetman One of the following thermal cyclers from Applied Biosystems GeneAmp PCR System 9600 GeneAmp PCR System 9700 GeneAmp PCR System 2400 DNA Thermal Cycler 480 Note If the GeneAmp PCR System 2400 or DNA Thermal Cycler 480 will be used to denature your samples you will need the appropriate materials e g tubes tray and so forth for these thermal cyclers You will also be required to transfer the samples to either the 96 or 48 well sample tray to load the samples onto the 310 Genetic Analyzer Consumables Sequencing application kit such as the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTag9 FS ABI PRISM 310 10X Genetic Analyzer Buffer with EDTA One of the following polymers POP 6 Performance Optimized Polymer with Template Suppression Reagent TSR POP 4 Performance Optimized Polymer Hi Di formamide if using POP 4 Matrix standard see page 6 25 Two 4 0
123. he virtual filter set that corresponds to the experiment s dye set by choosing a module file when setting up the run The virtual filter set s letter is in the module file name For example data is collected from BigDye Terminator sequencing samples using Virtual Filter E To run BigDye Terminator samples choose a module file such as SeqPOP6 1mL E md4 Some portion of a dye s emission profile may fall on a virtual filter other than the one intended to collect its emission maximum The dyes in each dye set are selected to have widely spaced emission maximums to minimize overlap of the emission profiles on the CCD array However overlap still occurs to some extent Matrix Files The spectral overlap between colors on the CCD array is corrected by a matrix file in the software The matrix file is generated from a separate matrix run and contains information about how much of the collected light falling on a filter is due to the intended light emission and how much is contaminating light When setting up the run you select a matrix file to interpret the data and correct for spectral overlap One matrix file can be used for all runs sharing the same conditions If run conditions are changed a new matrix file must be generated using the new run conditions Changes in run conditions affect dye fluorescence and the amount of spectral overlap Compatibility of The following dyes cannot be used in the same sample because there is no virtual Dye
124. hen 2 then 1 By telephone from any other country See the specific region under To Contact Technical Support by Telephone or Fax under Technical Support For chemicals not manufactured or distributed by Applied Biosystems call the chemical manufacturer Instrument Safety Safety labels are located on the instrument Each safety label has three parts Labels 1 6 AboutThis Guide Asignal word panel which implies a particular level of observation or action e g CAUTION or WARNING If a safety label encompasses multiple hazards the signal word corresponding to the greatest hazard is used A message panel which explains the hazard and any user action required A safety alert symbol which indicates a potential personal safety hazard See the ABI PRISM 310 Genetic Analyzer Site Preparation and Safety Guide for an explanation of all the safety alert symbols provided in several languages About Waste Profiles About Waste Disposal Before Operating the Instrument Safe and Efficient Computer Use A waste profile was provided with this instrument and is contained in the AB PRISM 310 Genetic Analyzer Site Preparation and Safety Guide Waste profiles list the percentage compositions of the reagents within the waste stream at installation and the waste stream during a typical user application although this application may not be used in your laboratory These profiles assist users in planning f
125. hen click Execute 3 Install a clean empty glass syringe with the syringe plunger at the bottom of its travel 4 From the Function drop down list select Syringe Down Move the syringe down gradually until it reaches the plunger 5 In the Status window go to the Injection drop down list and read the number in the Gel Pump At field Subtract 15 from that number 6 In the Manual Control window go to the Function drop down list and select Syringe Max Travel Type in the number you just calculated in step 5 then click Execute 7 Record this value on the Calibration sticker on the left side door Note If you install a syringe of a different size you must input the max travel value for that syringe size Homing the Syringe Home the syringe after Rebooting the instrument Changing the syringe max travel When prompted by the instrument To home the syringe Step Action 1 Open the Data Collection software 2 Open the Manual Control window 3 From the Function drop down list select Syringe Home then click Execute Manual Control x Function 108 Value Range EXTUS autosampler Present Tray 7 No Value No Range Electrophoresis Set Voltage Laser Set Power Laser Standby Temperature Set The 310 Genetic Analyzer homes the syringe 3 38 Operating the Instrument Hardware Handling and Each syringe is shipped with the plunger installed in th
126. hromatography BioCAD SPRINT VISION and INTEGRAI Workstations and POROS Perfusion Chromatography Products Expedite Nucleic Acid Synthesis Systems 1 800 899 5858 press 1 then press 3 1 800 899 5858 press 1 then press 4 1 800 899 5858 press 1 then press 5 1 508 383 7855 1 508 383 7855 1 508 383 7855 Peptide Synthesis Pioneer and 9050 Plus Peptide Synthesizers 1 800 899 5858 press 1 then press 5 1 508 383 7855 PNA Custom and Synthesis FMAT 8100 HTS System CytoFluor 4000 Fluorescence Plate Reader 1 800 899 5858 press 1 then press 5 1 800 899 5858 press 1 then press 6 5 1 508 383 7855 1 508 383 7855 To Contact Technical Support by Telephone or Fax Outside North America Product Product Area Chemiluminescence Tropix Telephone 1 800 542 2369 U S only or 1 781 271 0045 Fax 1 781 275 8581 LC MS Applied Biosystems MDS Sciex 1 800 952 4716 1 508 383 7899 a 5 30 AM to 5 00 PM Pacific time b 8 00 AM to 6 00 PM Eastern time c 9 00 AM to 5 00 PM Eastern time To contact Applied Biosystems Technical Support or Field Service outside North America use the telephone or fax numbers below Region Telephone Fax Eastern Asia China Oceania Australia Scoresby Victoria 61 3 9730 8600 61 3 9730 8799 China Beijing 86 10
127. ibration dot on the front of the tray platform with the capillary Center the end of the capillary on the x y calibration point Almost touch the z calibration point with the end of the capillary Note See the diagram under Calibration Points on page 3 4 7 Click Set to save the calibration value Repeat for the rear calibration point and click Set Follow the instructions on the screen to complete the calibration Autosampler Calibration Test The electrode and capillary must be immersed in the sample during electrokinetic injection Minimum sample volume is 10 uL Microcentrifuging the sample briefly to bring it to the bottom may be necessary Current problems and low signal problems can result if the electrode and capillary do not make contact with the sample Test manually that the autosampler calibration is allowing the capillary and electrode to make contact with your samples To test the autosampler calibration manually Step Action 1 Put an empty tube in position A5 of the autosampler Open the Data Collection software if not open already From the Window menu select Manual Control From the Function drop down list select Autosampler to Position Use the Autosampler to Position function to send the autosampler to position 5 Execute the Autosampler Up function NIO or A OJN Use the Autosampler Z Max Travel function to lower capillary and electrode into t
128. ich the serial cable from the instrument is connected 4 26 Using the 310 Data Collection Software Dye Indicators The Dye Indicators preferences allows you to change Preferences Dye Code Dye Color Plot Color GeneScan Injection List Defaults Sequence Injection List Defaults Folder Locations File Names Sequence Sample Sheet Defaults Preferences x GeneScan Sample Sheet Defaults General Settings Dye Indicators Collection Info B Bl Blue lo ll Green gt Green fy E yelow gt MMM Black JR B gt Res o EJ orange a Orange Reset to factory Settings Set the following dye indicator preferences Preference Description Code A single letter usually related to the dye s representational color or its name To change the dye code type a new letter in the code box Dye Color The color displayed on the GS sample sheet to represent fluorescence from that dye To change the color used to represent a dye select a new color from the drop down list Plot Color Color displayed on the electropherogram to represent fluorescence from that dye These are the colors displayed in the Raw Data window To change the color used to represent a dye select a new color from the drop down list Reset to factory Settings Click to set all the codes and colors back to their factory settings Using the 310 Data Collection Software 4 27
129. ided will result in death or serious injury This signal word is to be limited to the most extreme situations NUNNA CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Readand understand the material safety data sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Chemical Waste F WNIEXLMDC CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems Hazard Warning Site Preparation and Safety Guide About MSDSs instruments are potentially hazardous and can cause injury illness or death Read and understand the material safety data sheets MSDSs provided by the manufacturers
130. igh primer concentrations promote nonspecific annealing If you are not using AmpliTaq Gold DNA Polymerase consider performing Hot Start Technique Increase annealing temperature in 2 to 5 C increments Decrease annealing and or extension times Increase primer length Perform a second amplification with nested primers Perform Touchdown PCR Primer dimer and primer oligomer artifacts Check primers for 3 complementarity Design longer primers Reduce primer concentration Reduce number of PCR cycles Raise the annealing temperature in 2 to 5 C increments Increase amount of target DNA Incomplete restriction and or ligation if performing AFLP Repeat restriction and or ligation If performing AFLP too much DNA in reaction so that insufficient adaptor is present Use the recommended amount of template DNA Problems with Extra Peaks continued Observation Possible Causes Recommended Actions Extra peaks with PCR run contaminated with a Decontaminate equipment pattern Amplicons tools and working area b Check disposables and reagents as potential contaminated source Presence of split Partial nontemplate addition of an Add the correct amount of peaks differing in extra nucleotide usually Mg to the reaction mix size by one base adenosine to the blunt end of the pair GeneAmp PCR product Note High Mg
131. illary Replace the capillary Poor quality water in buffer solutions Remake buffer with fresh distilled deionized water Old defective or incorrectly made buffer or polymer Replace buffer or polymer Corrupted firmware Resend firmware Problems with Current continued Observation Possible Causes Recommended Actions Low current Small bubble in capillary blocking current flow Rerun module Plugged broken or nonconducting capillary Replace the capillary Poor quality water in buffer solutions Remake buffer with fresh distilled deionized water Old defective or incorrectly made buffer or polymer Replace buffer or polymer Fluctuating current Too little buffer in anode buffer reservoir Replenish buffer reservoir Small bubble in capillary blocking current flow Rerun module Broken or cracked capillary Replace the capillary Arcing to conductive surface on the instrument a Clean the heat plate electrode and autosampler b Ensure that the ambient temperature is between 15 and 30 C and the humidity is below 80 Check for excessive condensation on the instrument Current is normal at beginning of run and then decreases rapidly over the next several minutes Loss of anode buffer capacity Replace the buffer Overloaded capillary a Use TSR or formamide b Decrease the electrokinetic injecti
132. iments For detailed information about preparing your samples you will need to refer to the documentation for the chemistries and thermal cycler you are using the Automated DNA Sequencing Chemistry Guide and the Automated DNA Sequencing Chemistry Guide Setting Up Fragment Analysis Experiments This chapter describes how to set up the 310 Genetic Analyzer for a fragment analysis experiment Note This chapter provides general information for preparing fragment analysis experiments For detailed information about preparing your samples you will need to refer to the documentation for the chemistries and thermal cycler you are using and the GeneScan Chemistry Reference for the ABI PRISM 310 Genetic Analyzer Troubleshooting This chapter provides troubleshooting information for the 310 Genetic Analyzer Appendix A Limited Warranty This appendix provides warranty information about the instrument 1 2 AboutThis Guide Chapter Content Appendix B Consumables This appendix provides part number information about the consumables used by the 310 Genetic Analyzer About the Quick The Quick Reference Card P N 4317590 for the 310 Genetic Analyzer provides Reference Card information for performing a run as well as maintenance recommendations Related Documents This guide contains references to the following documents Document Part Number ABI PRISM 3
133. in solution Note You can store the capillary on or off the 310 Genetic Analyzer For more information see Storing the Capillary Off the Instrument on page 3 20 or Storing a Capillary On the Instrument on page 3 20 4 Close the instrument doors About a Long Term A long term shutdown is appropriate when you want to turn off the 310 Genetic Shutdown Analyzer for more than 2 days You may need to refer to the following procedures Procedure See page Removing the Capillary for Long term Storage 3 21 Removing the Gel Block 3 28 Removing Syringes 3 37 Cleaning the Gel Block 3 27 Cleaning the Syringe 3 35 Executing Functions 4 7 Starting a Module 4 7 3 44 Operating the Instrument Hardware Performing a To perform a long term shutdown Long Term Shutdown Step Action 1 Remove the capillary syringe and pump block 2 Wash the syringe and pump block 3 Reinstall the pump block and the capillary 4 Fill the syringe with distilled and deionized water and attach it to the pump block 5 Flush the capillary by running the Run Seq Fill Capillary module or the Syringe Down function in Manual Control The polymer in the capillary will be flushed to the waste tube on the autosampler Press the Tray button to present the autosampler Remove all chemistry and samples from the autosampler 8 Wipe the autosampler trays drip tray and elec
134. in water vials or buffer Change water and buffer vials regularly Syringe empty or incorrect Syringe Max Travel value Fill syringe if necessary and recalibrate Syringe Max Travel value Capillary too short Increase capillary length Note Increase electrophoresis time accordingly Ethanol contamination DNA sequencing Remove ethanol by evaporation Peaks trail or shoulder into next peak Template not accurately quantitated DNA sequencing Quantitate template Refer to the sequencing kit protocol for appropriate DNA quantities False stop large peak in all four colors in dye primer sequencing Secondary structure a Denature at 95 C for 5 minutes b Try dye terminator sequencing Failure to separate transformed and nontransformed vectors Use proper techniques to isolate transformed vector Primer dimer formation during PCR amplification Use different primers For more information see the following user manuals GeneScan Chemistry Reference for the ABI PRISM 310 Genetic Analyzer Automated DNA Sequencing Chemistry Guide Troubleshooting 8 17 Problems with Poor Amplification 8 18 Troubleshooting Problems with Poor Amplification Observation Possible Cause Recommended Actions Faint or no signal from sample DNA and from positive control Insufficient injection or a mispositioned capillary Check that
135. indow represent the colors by which the data is displayed These colors are set in the Dye Indicators preferences see Dye Indicators Preferences on page 4 27 When you click one of the buttons the line of data represented by that color is hidden and the button turns white To restore the line of data click the button again The colors in the real time data display differ from the colors in the display of data after analysis The Data Collection software defines specific areas on the CCD camera from which to collect light intensities These areas correspond to different wavelengths of light The result is similar to using a physical filter to separate the light wavelengths This is referred to as a virtual filter since no physical filtering hardware is used On the real time display the Raw Data window the Data Collection software displays these intensities color coded according to wavelength Blue green yellow and red in that order represent the wavelengths of the dye 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 relative wavelengths of the dyes being detected A virtual filter set represents the relative wavelengths of the dye set as blue green yellow and red regardless of the absolute value of the wavelengths in nanometers The colors do not represent actual wavelengths They represent the relative w
136. ing Experiments 6 27 To make a matrix file from matrix standards continued Step Action 3 From the Select Sample File dye X for Matrix Generation Look in 3 AppliedBio amp Ek r e 137 C MatrisMakerSC 377 96 C Seg nal 213774 CJ Shared L GelProcessor Ss GeneScan File name l Files of type sto mw Cancel For dye 1 browse to the dR6G file In this example it should be located in the Runs folder Click Open 4 Repeat step 3 for all four dyes Fill in the Select Sample File dye X for Matrix Generation dialog boxes using the following table dye X Sample File dye 1 dR6G dye 2 dTAMRA dye 3 dROX dye 4 dR110 Once the fourth file has been selected the Matrix Maker will compute the matrix Note The information in the table is for this example 5 When the Matrix Maker has completed computing the new matrix file the following window will appear Matrix Produced c 2 84 Save As 2 x Save in amp Matix amp Ek a BDT 2 Matrix mtx a jz Ponce 6 93 Matrix mtx a BDT 3 Matrix mtx is TestMatrix mtx a bdt 4 mATRIX mt a TestMatrixSdye mtx BDT STD Matrix mtx a Bogus Matrix mtx a BogusMatrixSdye mtx File name Save as type eus gm Cancel a Type in a file name b Click Save 6 28 Setting Up DNA Sequencing Experiments If You Get an Error f you get an error message and the software will
137. instrument 6 Transfer the samples to 0 5 or 0 2 mL sample tubes and cover with a tube septum Note You must use tube septa to prevent evaporation of samples especially if samples are put in the autosampler more than 6 hours before analysis The minimum sample volume per tube is 10 uL IMPORTANT If using 10 uL sample volumes it is extremely important that you calibrate the autosampler tray see Calibrating the Autosampler on page 3 4 You can use a single sample for more than one analysis Either inject out of one sample tube repeatedly or split the sample into several tubes Setting Up DNA Sequencing Experiments 6 9 Preparing a Portion Occasionally you may want to prepare only a portion of a sequencing reaction mixture of a Reaction for analysis on the 310 Genetic Analyzer and reserve the rest of the sample for Mixture for Analysis analysis later or elsewhere 7 Ne eh CHEMICAL HAZARD Template suppression reagent may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To prepare a portion of a sequencing reaction mixture Step Action 1 Add 6 uL of TSR to the dried DNA sequencing reaction Vortex to dissolve the extension products Heat the sample for 1 minute at 95 C to ensure denaturation Transfer 2 uL of the sample to 10 uL of TSR in a sample tube Cov
138. ion 00 0 eee eee ee eee 2 19 Electrophoresis 3 eto Hehe PAGES Ca a VEDO eS 2 19 Sample Fluorescence gnai cw ES pe Be RONDE E ES en d E S NES 2 19 Detection eer urs be LEA ee en RU areae e cbe de 2 20 D ta Collection is et eo Ee et oe ecb S RR Meg tO gape babe dee ep v 2 20 Virtual Filters and Dye Sets sse soos isese eR dk Ree RR Ra 2 20 Matrix Eill s oe a a RESUME NOTOS REN AS ARAL e 2 20 Compatibility of Dyes and Virtual Filters eee 2 20 Color Display of Data i i e R RE REP ERE ES 2 21 Virtual Filters and Associated Applications 00 0 0 eene 2 21 Color Guide for DNA Sequencing Applications eese 2 21 Color Guide for Fragment Analysis Applications 0 000000 2 22 3 Operating the Instrument Hardware Introduction cti e Rr SEC at CR ULIS Pie ee ee EE E etn ME 3 1 In This Ch pter 2 eoe eru BR eI E AU RUE RP I e 3 1 Preparing the 310 Genetic Analyzer fora Run 2 0 cece ee cee eee eee 3 2 Summary of Procedures lt iis 6505 ele Sad a Rb e EA RR RR RR Ee 3 2 What to DoNextz tive owes cxt eb proe aa tee eas VI 3 2 Section Procedures for Setting Up and Maintaining the Instrument 3 3 In This Sections oi x Ee Re RR RR RR ER AUS ROUEN 3 3 Autosampler s c v ere 64 Wee DESC VAR Sa Bee t requi REM TEE 3 4 Cleaning the Autosampler leleeeeeee e 3 4 Calibrating the Autosampler sseeeeeeeeeeee e 3 4 Homing the Autosampler 0
139. is chapter ABI PRISM9 310 Data Collection Software v 3 0 or higher One of the following data analysis software applications ABI PRISM Sequencing Analysis Software v 3 7 or higher ABI PRISM SeqScape Software v 1 0 or higher Summary of To perform a sequencing experiment on your samples using the 310 Genetic Procedures Analyzer do the following Step Procedure See Page Set up the 310 Genetic Analyzer as described in Chapter 3 Operating the Instrument Hardware Prepare the matrix standards if necessary Prepare the samples Load the samples Prepare a sample sheet using the created matrix file Prepare an injection list Start the sample run Create the matrix file if necessary 6 8 Setting Up DNA Sequencing Experiments Analyze the data Denaturing and Loading Samples About the Sample Preparation Reagents About TSR and Samples in TSR Preparing the TSR Sample Mix Minimum Sample Volume The following procedures are for preparing the samples using POP 6 sequencing protocols which use template suppression reagent TSR as the denaturant Note If you are using POP 4 sequencing protocols you will be using highly deionized formamide Hi Di formamide as the denaturant Refer to the POP 4 Polymer Sequencing Protocols for the 310 Genetic Analyzer User Bulletin for information about preparing the samples using formamide CHEMICAL HAZARD Formamide i
140. iscretion when adding software programs Although the hard disk is extremely reliable it is still subject to the whims of fate and power Always back up your computer s hard disk so you do not lose the data you have spent hours collecting and analyzing Keep the backups in a safe place When you use the Data Collection software and analysis programs regularly a large number of data files may accumulate on your hard disk and consume available disk space Make copies of the data files you use infrequently and remove the originals from your hard disk to reclaim storage space for future work Whenever files are written to the hard disk opened and rewritten their physical location on the disk changes This fragments the disk If a significant amount of fragmentation occurs the system runs slowly and files may be lost Protect your data by running a disk optimizer program at least once a month Keep an anti viral software application loaded on your hard disk and use it to inspect your hard disk either when you start up or shut down at least once a day Viruses can simply be annoying but they can also in the worst case destroy all information stored on your hard disk Use the computer only for 310 Genetic Analyzer software Prevent program conflicts before they occur Do not load games or other software programs onto your hard disk Do not use any software or games this includes custom startup sounds and graphics other than those
141. ix file using the GeneScan Analysis software 7 22 Setting Up Fragment Analysis Experiments Preparing Matrix Standards About Matrix Standards You must run matrix standards and create a matrix file the first time you use a new chemistry or change the run conditions Do not prepare matrix standards more than 2 hours in advance To ensure reproducibility of results for all samples prepare the matrix standard mix using the 12 1 ratio of reagents stated in the procedure below IMPORTANT Do not add size standard mix to the matrix standard samples The following are the matrix standards used for fragment analysis Matrix Standard Set DS 02 for filter set E5 dR6G dR110 dTAMRA dROX LIZ Matrix Standard Set DS 33 for filter set G5 6 FAM VIC NED PET LIZ Matrix Standard Set DS 30 6 FAM HEX NED ROX use Fluorescent Amidite Standards kit NED Fluorescent Amidite Standards 6 FAM HEX TET ROX TAMRA NED matrix standard VIC matrix standard Matrix Standard Set DS 32 for filter set F 5 FAM JOE NED ROX Matrix Standard Set DS 34 6 FAM TET HEX TAMRA use Fluorescent Amidite Standards kit for filter set C Note For part number information see Appendix B Consumables Preparing Matrix FNYENIN CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and Standard Samples may cause irritation to the eyes skin and respiratory tract It may caus
142. k box then click Deliver Selected Documents Now to have the document PDF format e mailed to you Note There is a limit of five documents per fax request but no limit on the number of documents per e mail request To Obtain Customer To obtain Applied Biosystems training information Training Information Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page then click Training About This Guide 1 13 Overview of the 310 Genetic Analyzer Introduction In This Chapter Topics in this chapter include the following Topic See Page Section Description of the Instrument 2 3 Front of the Instrument 2 4 Back of the Instrument 2 5 Behind the Doors of the Instrument 2 7 Section Description of the Computer and Software 2 13 Computer 2 14 Software 2 15 Section Theory of Operation 2 17 What the 310 Genetic Analyzer Does 2 18 Principles of Operation 2 19 Overview of the 310 Genetic Analyzer 2 1 About This Chapter This chapter will help you identify parts of the ABI PRISM9 310 Genetic Analyzer and understand how it works The 310 Genetic Analyzer is a system composed of instrument hardware a computer several types of software and consumables For More For information about the procedures for operating the 310 Genetic Analyzer see Information Chapter 3 Operating the Instrument Hardware For i
143. k the raw data for excessive noise The ideal baseline falls at or below about 2068 Use the following table to help determine whether the capillary is positioned correctly If Then the baseline is too high remove the capillary and clean the capillary window with a lab wipe and dampened ethanol there is a single blue line at 8000 orat the capillary window is not positioned the bottom of the window in the detector Operating the Instrument Hardware 3 19 Removing and Storing a Capillary On the Instrument Storing the IMPORTANT Leave the power off when storing the capillary with this method The Capillary autosampler moves up when the power is turned on To store the capillary on the instrument Step Action 1 Run the Seq Fill Capillary module in Manual Control to remove any sample still in the capillary Turn off the instrument The autosampler drops when the power is turned off Remove the thermal tape holding the capillary to the heat plate Lower the end of the capillary into the buffer vial in the autosampler c1 B OJIN Tape the capillary in this position and close all the doors Before Turning Power On IMPORTANT When a capillary has been stored on the 310 Genetic Analyzer you must reposition the capillary before turning power back on If you do not reposition the capillary the autosampler will break it when the instrument is turned on
144. l not affect the configuration of the instrument It is for record keeping purposes only Operator The name you enter here will appear on printed data Tube amp Sample Name The sample s name and position in the autosampler Module The file that contains the specific functions executed to process samples Inj Secs The duration of the injection in seconds Inj kV The voltage during the injection in kilovolts Run kV The voltage during the run in kilovolts Run C The temperature of the heat plate during the run in degrees Celsius Run Time The duration of the data collection in minutes This is less than the total run time Finish Time Displays the time at which raw data collection for the sample finished Note You may need to scroll to see the Run Time and Finish Time columns 6 16 Setting Up DNA Sequencing Experiments Editing Injection Lists About Electrokinetic Injection Voltage and Time The following are common things you may want to edit in an injection list Change the order of samples Addarow to the injection list Run a sample more than once Change the module for a sample See Chapter 5 Injection Lists for more information about the above procedures Change Run Parameters After a module is selected in the injection list the following fields are automatically filled in with information from the module Injection time Injection voltage Run voltage
145. lace the computer or printer the following table lists the minimum specification Item Specification CPU 733 MHz Intel Pentium IIl processor Monitor Color monitor 17 inch display or larger Disk drive Hard disk with a minimum of 2 GB storage preferably 10 GB Memory 256 MB random access memory Printer Color PostScript printer Operating system software Microsoft Windows NT 4 0 Service Pack 5 English version RAM Requirements RAM requirements for software applications are Software RAM Required ABI PRISM 310 Data Collection 96 MB Software ABI PRISM Sequencing Analysis 10 MB Software and basecallers ABI PRISM SeqScape Software 7 MB ABI Prism GeneScan Analysis 10 MB Software ABI PRISM GeneMapper Software 38 MB 2 14 Overview of the 310 Genetic Analyzer Software Overview Software for Data Collection The 310 Genetic Analyzer comes with software programs that perform Raw data collection Data analysis Software Purpose Data Collection software Controls monitors and collects data from the 310 Genetic Analyzer instrument Sequencing Analysis software Analyzes raw sequencing data and calls bases de novo sequencing SeqScape software Compares DNA sequences Used for a variety of mutation detection applications GeneScan Analysis software Sizes and quantifies DNA fragments GeneMapper software Compares DNA fragment
146. lass capillary filled with polymer An anode electrode at the other end of the capillary is immersed in buffer A portion of the sample enters the capillary as current flows from the cathode to the anode This is called electrokinetic injection The end of the capillary near the cathode is then placed in buffer Current is applied again to continue electrophoresis When the DNA fragments reach a detector window in the capillary coating a laser excites the fluorescent dye labels Emitted fluorescence from the dyes is collected by a CCD camera The software interprets the result calculating the size or quantity of the fragments from the fluorescence intensity at each data point The software programs available for fragment analysis are GeneScan Analysis software GeneMapper software 2 18 Overview of the 310 Genetic Analyzer Principles of Operation Sample Labeling Automated Sample Handling Electrokinetic Sample Injection Electrophoresis Sample Fluorescence The samples are labeled using a dye set comprised of dyes that fluoresce at different wavelengths The dyes might label either different bases in the DNA sequence or different fragments in an experiment Fluorescently labeled samples are placed in the autosampler The cathode electrode and one end of a polymer filled capillary are located near the autosampler The autosampler brings samples in the sample tray successively into contact with them A short period of ele
147. le column in the injection list The reference table on pages 6 20 and 6 21 will help you to select the appropriate module If a custom module is necessary see Editable Parameters on page 4 14 The dye set primer files also known as mobility files are selected in the Dye Set Primer column in the injection list They contain information that adjusts the sample data collected during electrophoresis for The dye set Mobility of the primers or terminators The dye set primer file contains mobility correction data for the sample Note that the dye set primer file choice must be consistent with the sequencing reaction chemistry polymer and filter set Select the dye set primer file through the sample sheet The reference table on pages 6 20 and 6 21 will help you select the appropriate dye set primer file Note Dye Set Primer files cannot be edited The table on pages 6 20 and 6 21 lists the dye set primer mobility files and the modules for DNA sequencing runs Setting Up DNA Sequencing Experiments 6 19 gour A ede LW eAda rdOd0l da ueej dog x uo 7p pdOdOLE ypur3 1u L bespideurg qour LA LINLZ e dardodoLeda qour LA eHe LIWN e dardOdOlLeda ueej dog x uo 7p vdOdOLE ypur3 qurL bespisrd qour LMELINLZ eE agirdOd0Ledd V dOd qour esJe ege LW 19Sdg 9dOd Ld 10 qour LAM eu LA e ag edodoreda qourt LN Lz iesagiedod1a ueejb dog xu
148. les See modules run parameters electrophoresis temperature DNA sequencing 6 18 fragment analysis 7 17 electrophoresis time DNA sequencing 6 18 fragment analysis 7 17 electrophoresis voltage DNA sequencing 6 18 fragment analysis 7 17 voltage and time DNA sequencing 6 17 fragment analysis 7 17 run status 4 11 to 4 13 Electorphoresis History window 4 13 Log file about 4 11 to 4 12 running voltage setting parameters DNA sequencing 6 18 fragment analysis 7 17 S sample fluorescence 2 19 Index 5 sample handling automated 2 19 Sample Info column fragment analysis 7 11 Sample Name column DNA sequencing 6 12 fragment analysis 7 10 sample sheet defined 6 11 7 9 DNA sequencing Collection Name column 6 13 Comments column 6 13 Dye Set Primer column 6 12 filling out 6 11 to 6 14 to 7 12 Matrix column 6 12 Sample Name column 6 12 exporting 6 14 fragment analysis Collection Name column 7 10 Color column 7 11 Comments column 7 11 filling out 7 9 to 7 11 Sample Info column 7 11 Sample Name column 7 10 Std column 7 11 GeneScan defaults preferences 4 23 GeneScan example 7 10 importing 6 14 modify an existing sample sheet DNA sequencing 6 13 fragment analysis 7 12 preferences 4 22 to 4 23 Sequencing defaults preferences 4 22 sample trays 48 well numbering 3 31 96 well inserting in autosampler 3 34 numbering 3 32 preparing 3 33 loading samples DNA sequencing 6 10 fragment analysis 7 8 part numbers B 2 types 3 31 sam
149. lification Note For fragments smaller than 130 bp use the Amicon Centricon 30 column instead Add bovine serum albumin BSA to the PCR reaction mixture Sample DNA is degraded If possible evaluate the quality of DNA sample by Using the QuantiBlot Human DNA Quantitation Kit Running an agarose yield gel If DNA is degraded 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 For more information see the following GeneScan Chemistry Reference for the ABI PRISM 310 Genetic Analyzer Automated DNA Sequencing Chemistry Guide Poor yield for multiplex PCR Incorrect thermal cycling parameters Add a 2 minute down ramp time to thermal cycling profile between the denaturation and annealing stages Note For multiplex PCR a short down ramp time is not necessarily optimal Problems with Poor Amplification continued Observation Possible Cause Recommended Actions 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
150. list Similarly when a 4 dye sample sheet is imported only 4 dye modules are available c Leave the remaining parameters at their default settings In the Matrix File column select the name of the matrix being used Check the Auto Anlz box to launch the GeneScan Analysis software automatically Check the Auto Prt box to print the data automatically 7 14 Setting Up Fragment Analysis Experiments GeneScan Injection Drag the markers at the top of columns to change their width Fields in the GeneScan injection list form Field Description Sample Sheet Displays the selected sample sheet Page icon Opens the selected sample sheet Length to Detector The distance between the end of the capillary that is in the sample and the capillary window The length you enter in the Injection List will not affect the physical configuration of the instrument It is for record keeping purposes only Operator The name you enter here will appear on printed data Tube amp Sample Name The sample s name and position in the autosampler Module The file that contains the specific functions executed to process samples Inj Secs The duration of the injection in seconds Inj kV The voltage during the injection in kilovolts Run kV The voltage during the run in kilovolts Run C The temperature of the heat plate during the run in degrees Celsius Run Time The duration of the data co
151. llection in minutes This is less than the total run time Matrix file The matrix file to be used for each sample file Auto Anlz Determines if raw data is analyzed automatically by the GeneScan Analysis software Analysis Parameters The parameters file to be used Size Standard The size standard file to be used Auto Prt Determines if analyzed data is printed automatically Setting Up Fragment Analysis Experiments 7 15 Editing Injection The following are common things you may want to edit in an injection list Lists 4 Change the order of samples Add a row to the injection list Runasample more than once Change the module for a sample See Chapter 5 Injection Lists for more information about the above procedures Change Run Parameters After a module is selected in the injection list the following fields are automatically filled in with information from the module Injection time Injection voltage Run voltage Run temperature 9 9 9 Run time You can edit these parameters by selecting a field and typing in a new value This changes the run conditions for one run It does not permanently alter the module For information about editing a module see Editing Modules on page 4 14 Change Sample Sheet Items from the Injection List The following files on a sample sheet can be changed after viewing the sample sheet from the injection list Analysis parameters Size s
152. ltage for strength allows shorter run times but reduces base calling accuracy DNA Sequencing For Sequencing Samples Using The Standard Running Voltage Is POP 4 Standard 240 volts cm For a 47 cm capillary this translates to 11 3 kV The current at this voltage is 5 pA Temperature is 50 C The time required for a 525 base fragment to reach the detector window is approximately 32 minutes with these run conditions POP 4 RAPID 320 volts cm For a 47 cm capillary this translates to 15 kV The current at this voltage is 7 pA Temperature is 50 C The time required for a 425 base fragment to reach the detector window is approximately 20 minutes with these run conditions POP 6 and long read sequencing 200 volts cm For a 61 cm capillary this translates to 12 2 kV The current at this voltage is 4 to 6 pA The time required for a 600 base fragment to reach the detector window is approximately 120 minutes with these run conditions POP 6 and RAPID sequencing 320 volts cm For a 47 cm capillary this translates to 15 kV The current at this voltage is 5 to 8 pA The time required for a 400 base fragment to reach the detector window is approximately 36 minutes with these run conditions Note The required times listed in the above table are approximations only The times can vary from instrument to instrument Electrophoresis The temperature limits of the heat plate are 60 C down to 5
153. lyzed files See Color Display of Data on page 2 21 for more information about color display in raw and analyzed data Setting Up DNA Sequencing Experiments 6 25 Preparing Matrix PNI CHEMICAL HAZARD Template suppression reagent may cause eye skin Standard Samples and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves To prepare matrix standard samples Step Action 1 For each of the four matrix standards mix in a sample vial 12uLofTSR 1 uL of matrix standard Label each vial with the base and dye Gently vortex the mixture for 3 to 5 seconds Store the mix at 2 to 6 C until ready to use Note If you are using POP 4 you will need to set up your matrix standard samples using Hi Di formamide See the POP 4 Polymer Sequencing Protocols for the 310 Genetic Analyzer User Bulletin Denaturing the You can denature the matrix standard samples in the thermal cycler with your other Matrix Standard samples refer to Denaturing and Loading Samples on page 6 9 or follow this Samples Procedure Step Action 1 Heat the sample for 2 minutes at 95 C 2 Chill the sample on ice 3 Hold on ice until ready to load in the autosampler 6 26 Setting Up DNA Sequencing Experiments Creating Matrix Files from Matrix Standards About This Example n this e
154. mL Genetic Analyzer buffer vials One 1 0 mL glass syringe Two septa for the 4 0 mL buffer vials One of the following capillaries For rapid sequencing ABI PRISM 310 Genetic Analyzer Capillary 47 cm 50 um i d labeled with a green mark For long read sequencing ABI PRISM 310 Genetic Analyzer Capillary 61 cm 50 um i d labeled with a pink mark Note Do not use sequencing capillaries labeled with a silver mark P N 401821 with POP 6 polymer Resolution will be unsatisfactory if these capillaries are used with POP 6 polymer Mineral oil for the DNA Thermal Cycler TC1 and the DNA Thermal Cycler 480 Setting Up DNA Sequencing Experiments 6 7 96 well tray 48 well tray One sample tray and related accessories One MicroAmp tray retainer set One box of 0 2 mL MicroAmp reaction tubes One MicroAmp base One adaptor Note f you are using a 9700 Thermal Cycler and you do not have the 96 well sample tray adaptor P N 4305051 then you will have to transfer the samples from the tray compatible with the 9700 after denaturation to the 96 well sample tray before placing it on the autosampler One MicroAmp full plate cover One Genetic Analyzer septa strip One Genetic Analyzer retainer clip 48 well sample tray 4 0 mL One box of 0 5 mL sample tubes One box of septa for 0 5 mL sample tubes Software Required The following software is required for the procedures described in th
155. making the matrix file Making a Matrix To make a matrix file form a sample File Step Action 1 In the Sequencing Analysis software a Go to the Sample drop down list b Select Make Matrix In the Input file and dye options window Input file and dye options X Enter number of sample files to use 1 4 5 4 Enter number of dyes 4 5 4 Cancel OK a Set the number of sample files to use to 1 b Set the number of dyes to 4 c Click OK 6 30 Setting Up DNA Sequencing Experiments To make a matrix file form a sample continued Step Action 3 From the Select Sample File for Matrix Generation Look in C AppliedBio El e Cre 377 C Seg nal 377 96 CJ Shared 3774 Ss GelProcessor GeneScan File name Files of type pes m Cancel a Browse to find the sequencing standard file It should be in a folder located on D AppliedBio 310 Runs b Click Open Once the file has been selected the Matrix Maker will compute the matrix 4 window will appear Matrix Produced Save As 27x Save in E Mai o E ei a BDT 2 Matrix mtx aa iz Ponce 5 93 Matrix mtx a BDT 3 Matrix mtx a OneFileM atrix mtx bdt 4 mATRIX mtx a TestMatrix mtx a BDT STD Matrix mtx a TestMatrixSdye mtx a Bogus Matrix mtx a BogusMatrixSdye mtx File name Save as type mts x Cancel a T
156. mal Cycler 9600 9700 will be used to denature your samples you will need the appropriate materials e tubes tray etc for these thermal cyclers You will also have to transfer the samples to either the 96 or 48 well sample tray to load the samples on the 310 Genetic Analyzer Consumables ABI PRISM 310 10X Genetic Analyzer Buffer with EDTA One of the following polymers POP 4 Performance Optimized Polymer GeneScan Polymer Hi Di formamide Matrix standard see page 7 23 One of the following size standards GeneScan 400HD ROX GeneScan 500 ROX GeneScan 500 LIZ GeneScan 120 LIZ Two 4 0 mL Genetic Analyzer buffer vials with cap adapters One 1 0 mL glass syringe or one 2 5 mL glass syringe Two septa for the 4 0 mL buffer vials ABI Prism 310 Genetic Analyzer Capillary 47 cm 50 i d labeled with a green mark Note Do not use GeneScan capillaries labeled with a yellow mark P N 401823 with POP 4 polymer Resolution will be unsatisfactory if these capillaries are used with POP 4 polymer Setting Up Fragment Analysis Experiments 7 5 One sample tray and related accessories 96 well tray One MicroAmp tray retainer set Two to ten 0 2 mL MicroAmp reaction tubes One MicroAmp base One adaptor Note If you are using a 9700 Thermal Cycler and you do not have the 96 well sample tray adaptor P N 4305051 then you will have
157. me To overwrite the original choose Save without renaming the sample sheet 3 Select the modified Sample Sheet when filling out the Injection List Setting Up DNA Sequencing Experiments 6 13 Importing Sample You can import data from tab delimited text files into the grids of the sample sheet Sheets Each row in the text file should contain the information for one row in the grid These data fields must also be in the same order as the columns of the grid Everything up to the first tab in the text file is imported into the first field in the grid To import files into a sample sheet Step Action 1 Create a new sample sheet 2 From the File menu select Import 3 In the dialog box that appears choose the name of the file containing the text you wish to import Click OK The corresponding text is imported into the sample sheet Exporting Sample Use the Export command to save the contents of a window grid for example from the Sheets sample sheet into a tab delimited text format This format is useful for exporting to most database word processing or spreadsheet programs To export information to tab delimited text format Step Action 1 Create a new sample sheet and fill in the necessary information From the File menu select Export Type a file name in the dialog box that appears 2 3 4 Click OK This creates a text file that can
158. mple sheet You can add new Injection List rows before or after an existing row in the injection list To fill in the fields for Tube amp 5 6 Injection Lists Sample Name and Module in a new row select from the drop down lists For all the other fields type a value You can add lines to the injection list before a run begins or while it is in progress however you cannot add lines above an injection that is running or already completed Adding One Row To add rows highlight a row select Insert from the Edit menu and a new row will be inserted above the highlighted row Adding Multiple Rows To add more than one row highlight the number of rows you want to add then select Insert For example if you want to add three new rows above row 2 select rows 2 3 and 4 then select Insert from the Edit menu Three new rows will be inserted above row 2 Adding a Row to the End of the Injection List To add a row at the end of the injection list select Add from the Edit menu Running a Sample More Than Once Changing Run Parameters Changing the Order of the Samples in a Run The injections are sequentially numbered To run a sample more than once add a row to the injection list Then use the drop down lists in the Tube amp Sample Name column to select the sample for more than one injection or copy and paste the entire injection on the new line As you select a module file for each injection the run parameters are filled in a
159. mples STE BEE GeneScan Sample Sheet 4Dyes m el Sample Hame sta Sample Info A1 1347 02 Mother none 1347 02 Mother 1347 02 Mother 1347 02 Mother 1347 10 Son none 1347 10 Son 1347 10 Son 1347 10 Son ai Matrix Standard 1 none Matrix Standard 1 Matrix Standard 2 none Matrix Standard 2 5 Matrix Standard 3 none Matrix Standard 3 Matrix Standard 4 none Matrix Standard 4 GeneScan sample sheet without matrix standard samples Eduntitied 1 Jol x GeneScan Sample Sheet 4Dyes Y Collection Name Cotor Std Sample Info 1347 15 Maternal Grandmother lt none gt 1347 15 Maternal Grandmother 1347 15 Maternal Grandmother 1347 15 Maternal Grandmother Size Standard 1347 02 Mother none 1347 02 Mother 1347 02 Mother 1347 02 Mother Size Standard 1347 01 Father none 1347 01 Father 1347 01 Father 1347 01 Father Size Standard 1347 10 Son nones 1347 10 Son 1347 10 Son 4 4347 10Son Size Standard Select 4 Dyes or 5 Dyes from the drop down list in the upper right corner In the Sample Name column enter the name of each of the samples In the Collection Name column select a collection name for the sample This is necessary only if you will be uploading the samples into the Sequence Collector software
160. n Applications Experiments Microsatellite Analysis Fluorescent genotyping for genetic linkage studies Paternity identification Forensic identification of samples Determination of loss of heterozygosity Microsatellite instability Trisomy analysis 9 9 9 9 Amplified Fragment Length Polymorphism AFLP Analysis Gene mapping using AFLP analysis Gene Expression Profiling Differential display Quantitative expression of gene products RNase protection assays Mutation Detection SNP Genotyping e 9 9 Single strand conformation polymorphisms SSCP Heteroduplex mobility assays HMA Mismatch cleavage Oligonucleotide ligation assays OLA Allele specific PCR 9 9 9 7 A Setting Up Fragment Analysis Experiments Performing a Fragment Analysis Experiment Materials Required To run a fragment analysis experiment you will need the following items For part number information for the consumables see Appendix B Consumables Lab Equipment Filter sterilized deionized water Disposable non powdered chemical resistant gloves Microcentrifuge or centrifuge adapted for spinning microtiter plates 96 well tray Small volume calibrated pipette and tips we recommend the Gilson Pipetman One of the following thermal cyclers from Applied Biosystems GeneAmp PCR System 9600 GeneAmp PCR System 9700 Note If the GeneAmp PCR System 2400 or DNA Ther
161. n 1 Remove the capillary if it is on the instrument 2 Unscrew and remove the cathode electrode thumbscrew Thumbscrew There are three holes in the electrode thumbscrew the capillary hole an inner electrode hole and an outer electrode hole Put the long arm of the cathode electrode in the inner electrode hole Electrode thumbscrew Outer electrode hole Inner electrode hole Capillary hole Electrode Put the short arm of the electrode in the outer hole Push the electrode down until it is flush with the top of the electrode thumbscrew Screw the electrode thumbscrew back onto the instrument N oci amp Recalibrate the autosampler after installing the electrode 324 Operating the Instrument Hardware Trimming the It may be necessary to trim a new electrode so that the end is flush with the stripper Electrode plate To trim the electrode Step Action 1 Open the Data Collection software if not open already 2 In the Manual Control window open the Function drop down list and select Home Z Axis Use the wire cutter P N T 6157 provided in the instrument packing kit Hold the cutters with the flat cutting face toward the top of the instrument Note The wire cutter provided in the instrument packing kit is a flush cutting wire cutter Do not use other types of wire cutter Cut the electrode flush with the lowe
162. n product termination and labeling occur simultaneously for all bases in one tube All methods use AmpliTaq DNA Polymerase FS The required polymer and reagents used vary Consult the protocol that comes with the Ready Reaction Kit Description of There are three levels of recommendation in the chemistry tables Chemistry Table Levels Level Description Recommended This chemistry is the best choice for most customers most of the time Satisfactory This chemistry will work Customers will get good results but there is a better choice of chemistry for the given application or platform Not This chemistry might work Recommen 3 ecommenged Some customers will get good results but others will not In most cases the results will be suboptimal in terms of readlength and or accuracy Setting Up DNA Sequencing Experiments 6 5 310 Chemistry The following table describes the chemistry options for DNA sequencing applications Options Table 310 Chemistry Options DNA Sequencing BigDye dRhodamine BigDye Standard Standard Application Terminators Terminators Primers Primers Terminators de novo Sequencing Recommended Satisfactory Recommended Satisfactory Satisfactory High Throughput de novo Sequencing Recommended Satisfactory Satisfactory Satisfactory Satisfactory Mid Low Throughput Comparative Sequencing Recommended Satisfactory Recommended R
163. ncorrectly made buffer or polymer Replace buffer or polymer Injection time too long broad peaks Decrease injection time Incorrectly prepared or degraded sample Prepare new sample Incorrect buffer formulation Check if buffer formulation matches protocol requirements Incorrect polymer composition Check if polymer composition matches protocol requirements Electrophoresis voltage too high Decrease electrophoresis voltage by as much as 4 kV Note Increase electrophoresis time accordingly Sample concentrated by evaporation leaving excess salt behind Do not concentrate sample by evaporation Use an Amicon Centricon 100 column if necessary Incomplete strand separation due to insufficient heat denaturation Make sure the samples are heated at 95 C for 5 minutes prior to loading onto autosampler Too much DNA in sample Dilute sample before adding to formamide Wrong capillary used for POP 4 or POP 6 runs Verify that you are using the correct capillary Problems with Peak Quality and Resolution continued Observation Possible Cause Recommended Actions Poor resolution continued Oil in sample from DNA Thermal Cycler 480 Carefully pipette PCR product without oil carryover Remove oil by organic extraction Poor quality water Use freshly autoclaved distilled deionized water Bacterial contamination
164. nd it will appear to the right of the command in the menu Using the 310 Data Collection Software 4 29 Injection Lists Introduction In This Chapter Topics in this chapter include the following Topic See Page About the Injection List 5 3 Working with Injection Lists 5 4 Things You Can Do During a Run 5 9 Injection Lists 5 1 About This Chapter Procedures Included in This Chapter Procedures Not Included in This Chapter 5 2 Injection Lists The information in this chapter is meant to supplement the injection list information in Chapter 6 Setting Up DNA Sequencing Experiments and Chapter 7 Setting Up Fragment Analysis Experiments This chapter describes additional procedures used for working with injection lists Creating an Injection List for DNA Sequencing To create an injection list for DNA sequencing see Preparing the DNA Sequencing Injection List on page 6 15 Creating an Injection List for Fragment Analysis To create an injection list for fragment analysis see Preparing the Fragment Analysis Injection List on page 7 13 About the Injection List Overview The injection list specifies the order for running samples how many injections are made from each sample and the module and running conditions for each injection The Run button in the injection list is used to begin a run Fill out the injection list in the ABI PRISM9 310 Data Collection Software to direc
165. nformation about the software used with the 310 Genetic Analyzer see the following chapters Chapter 4 Using the 310 Data Collection Software Chapter 5 Injection Lists Chapter 6 Setting Up DNA Sequencing Experiments Chapter 7 Setting Up Fragment Analysis Experiments For part number information about the consumables used with the 310 Genetic Analyzer see Appendix B Consumables 2 2 Overview of the 310 Genetic Analyzer Section Description of the Instrument In This Section This section covers the following topics Topic See Page Front of the Instrument 2 4 Back of the Instrument 2 5 Behind the Doors of the Instrument 2 7 Overview of the 310 Genetic Analyzer 2 3 Front of the Instrument Diagram Left door Right door Status ien indicators Doors The doors protect you from exposure to the instrument s laser and electrophoresis power supply The doors must be closed to begin a run The run interrupts if they are opened Status Indicators The status indicators are red amber and green lights The pattern of lights indicates the instrument s state Instrument Status LED Pattern Ready for use Steady green Running Blinking green End of run Steady green with blinking amber Paused door open or awaiting completion Blinking amber of self test Failure check the error log in the Data Steady red Collection software f
166. ng 3 47 troubleshooting with resets 3 46 types of resets 3 46 when to reset 3 46 shutting down 3 44 to 3 45 switching applications 3 50 turning on 3 43 turning on if capillary is installed 3 20 3 43 48 well tray sample numbering 3 31 96 well tray sample numbering 3 32 using septa strips with 3 32 A ABI PRISM 310 Data Collection Software See Data Collection software ABI PRISM 310 Genetic Analyzer See 310 Genetic Analyzer ABI PRISM GeneMapper Software See GeneMapper software ABI PRISM GeneScan Analysis Software See GeneScan Analysis Software ABI PRISM SeqScape Software See SeqScape software agarose gel using to evaluate sample 8 20 amber LED light status defined 2 4 amplification troubleshooting 8 18 to 8 21 analysis parameters file 4 26 analyzing data DNA sequencing 6 22 fragment analysis 7 20 autosampler 3 4 to 3 9 calibrating 3 4 calibrating calibration test 3 6 calibration keyboard equivalents 3 5 calibration points 3 4 cleaning 3 4 filling buffer reservoirs 3 8 homing 3 6 loading samples 3 9 numbering samples 3 31 to 3 32 power down warning 3 49 tray types 3 31 autosampler region parts of 2 11 B backing up files 3 51 base color assigned after analysis 4 9 buffer 3 10 correcting leaks 3 12 loading on autosampler preparing 3 10 preparing for single run 3 10 replacing when to 3 10 storing 3 10 3 8 C cables and power cords routing 3 42 calibration data recording 3 48 calibr
167. nge up and down Set the temperature of the heat plate Turn off the heat plate Set the autosampler Z buffer travel value Set the autosampler Z maximum travel value Make and send calibration files Set the CCD camera X and Y pixel positions Cold boot the instrument Set the syringe pump force value 9 9 9 9 9 9 9 9 9 9 9 9 9 9 94 94 9 94 9 9 Set the syringe maximum travel value 4 6 Using the 310 Data Collection Software Open the Manual To open the Manual Control window select Manual Control from the Window menu Control Window 4manual Control x Function Value Range Execut xecute autosampler Present Tray N No Range Module none z oi Executing Functions To execute a function Step Action 1 Select the function from the Function drop down list 2 Some functions require a value such as temperature in C or voltage in kV to work If the function you select requires a value enter it in the Value field 3 Click the Execute button to start the function Starting a Module To start a module Step Action 1 Select the module file you want to run from the Module drop down list Note The drop down list displays the contents of the Modules folder selected as the default in Folder Location preferences To use a module file in a folder other than the default folder select Other from the drop down list and locate the file Click the Start button to start the modul
168. ninterruptible power supply UPS if voltage spikes and power outages are a persistent problem Clean the electrode heat plate and autosampler often to avoid the build up of conductive debris that causes arcing Wiping with a water dampened lab wipe is sufficient Clean the gel block as often as recommended Polymer that dries in the gel block can be difficult or impossible to remove Protect the instrument from excessive dust Avoid twisting power cords around 310 Genetic Analyzer data communication cables Power cords can affect the performance of computer cables The distance over which data communications are reliable depends upon both networking protocol and the type and quality of the cable Connect the 310 Genetic Analyzer and the computer that is collecting data with no more than six feet of cable Use the cables provided with the instrument 3 42 Operating the Instrument Hardware Turning On the Genetic Analyzer If a Capillary Is To turn on the 310 Genetic Analyzer if a capillary is already installed Already Installed Step Action 1 Remove the capillary from the buffer tube Note The capillary is inserted in a buffer vial on the autosampler when it is stored on the instrument Thread the capillary through the electrode thumbscrew It should extend 0 5 mm beyond the electrode See Positioning the Capillary Near the Electrode on page 3 18 Turn on the instrument Check the LEDs on the fro
169. nnels for dried polymer which looks like white residue Rinse the block and its channels with distilled deionized water Force air through the channels using the plastic syringe or canned compressed air until the channels are dry Note This removes residual water from the gel block and fittings and ensures that the running polymer is not diluted Replace the gel block and buffer reservoir Operating the Instrument Hardware 3 27 Removing the Gel The gel block rides on two steel shafts It slides freely after a spring plunger in the Block block moves past an indentation on one of the steel shafts For a diagram of the inside of the instrument see Behind the Doors of the Instrument on page 2 7 To remove the gel block from the 310 Genetic Analyzer Step Action 1 Open the Data Collection software if not open already From the Window menu select Manual Control From the Manual Control window move the syringe drive up Remove the glass syringe and plastic syringe Remove the capillary Remove the buffer reservoir by gently twisting and pulling it straight down NIOJ OAI A OIN Remove the gel block by pulling it straight toward you 3 28 Operating the Instrument Hardware Installing the Gel To install the gel block on the 310 Genetic Analyzer Block Step Action 1 Verify that the gold electrode socket on the back of the block is dry
170. noise level increases so that bases cannot be called The table below shows the recommended quantities for each method Recommended DNA template quantity for each chemistry Cycle Sequencing Chemisiry Dye dRhodamine BigDye BigDye Template Primer Dye Terminator Terminator Primer Terminator PCR product 100 to 200 bp 2to5ng 1 to 3 ng 1 to 3 ng 210 5 ng 1 to 3 ng 200 to 500 bp 5 to 10 ng 3 to 10 ng 3 to 10 ng 5 to 10 ng 3 to 10 ng 500 to 1000 bp 10 to 20 ng 5 to 20 ng 5 to 20 ng 10 to 20 ng 5 to 20 ng 1000 to 2000 bp 20 to 50 ng 10 to 40 ng 10 to 40 ng 20 to 50 ng 10 to 40 ng 22000 bp 50 to 150 ng 40 to 100 ng 40 to 100 ng 50 to 150 ng 40 to 100 ng single stranded 150 to 300 ng 100 to 250 ng 50 to 100 ng 200 to 400 ng 50 to 100 ng double stranded 300 to 600 ng 200 to 500 ng 200 to 500 ng 200 to 800 ng 200 to 500 ng cosmid BAC 0 5 to 1 0 ug 0 5 to 2 0 ug not 300 to 600 ng 300 to 600 ng recommended genomic DNA not recommended 2 to 3 ug 6 36 Setting Up DNA Sequencing Experiments About Primer Design Primer Design The choice of primer sequence method of primer synthesis and approach to primer purification can have a significant effect on the quality of the sequencing data obtained in dye terminator cycle sequencing reactions Dye primer cycle sequencing kits include dye labeled primers that are already optimized and quantitated For troubleshooting information refer to Primer Problems on pa
171. nt The green light should be illuminated Turn on the computer Open the Data Collection software Open the Manual Control window select Syringe Home and click Execute OINI OIA Aa 0 In the Manual Control window a Select Autosampler Home X Y and click Execute b Select Autosampler Home Z and click Execute Calibrate the autosampler See Calibrating the Autosampler on page 3 4 Turning On the To turn on the 310 Genetic Analyzer Instrument Step Action 1 Turn on the instrument 2 Check the LEDs on the front The green light should be illuminated 3 Turn on the computer 4 Open the Data Collection software 5 Open the Manual Control window select Syringe Home and click Execute 6 In the Manual Control window a Select Autosampler Home X Y and click Execute b Select Autosampler Home Z and click Execute Operating the Instrument Hardware 3 43 Shutting Down the Genetic Analyzer About a Short Term Shutdown Performing a Short Term Shutdown A short term shutdown is typically for no more than 2 days and used to conserve power The key to a short term shutdown is keeping the capillary in solution This prevents polymer from drying in the capillary To perform a short term shutdown Step Action 1 Shut down the computer 2 Turn off the instrument The LED status lights will turn off 3 Store the ends of the capillary
172. nt has run for 4 days without any of the above occurring IMPORTANT The gel block can be irreversibly damaged if polymer dries in the channels It can be difficult or impossible to resolubilize dried polymer Clean the gel block as often as recommended It is especially important to clean the gel block if a syringe containing polymer was installed on the block for more than 1 week At room temperature sufficient urea decomposition occurs in 1 week to cause transient current increases during electrophoresis IMPORTANT Do not expose the gel block to any organic solvents Do not clean the gel block with organic solvents or boiling water How to Clean the Gel Block To clean the gel block Step Action 1 Remove the gel block from the instrument Open all valves and ports on the gel block Hold the gel block under warm running water to thoroughly rinse it 2 3 4 Force warm pure water through the channels with the 5 cc plastic syringe provided in the Basic Installation Kit Direct the flow of water through each channel in turn by sealing channel openings with plugs provided in the Basic Installation Kit Rinse each channel five times Note If a 30 cc syringe is available rinse each channel with it once This can be more convenient than rinsing five times with a 5 cc syringe Rinse the valves with warm pure water Note Soak valves that are coated with dried polymer Visually inspect the cha
173. nter the starting point for each file The Start At point should be after the primer peak If necessary Modify the default setting of 2000 Modify the Points value This is the number of points after the start point to be analyzed Click OK A successful matrix opens an untitled Matrix Values window with a 5x5 matrix of numerical values Reactions B G Y R IEEE 02252 0020s 00074 oo1581 oss 10000 53487 foosss oos oa208 05395 10000 fosss2 oo084 o221s oa127 o7422 10000 o0120 Joozs7 5 0560 fo 1so2 02544 10000 mr qma mm uh deu Note For a four dye set the window displays a 4x4 matrix of numerical values For a five dye set the window displays a 5x5 matrix of numerical values 7 26 Setting Up Fragment Analysis Experiments To make a matrix file continued Step Action 6 Use the Save As command to name and save the matrix file Choose a name that reflects the chemistry and run conditions Checking the Matrix Check the quality of the matrix by reviewing the Quality 4 values in the Matrix Values window Analyzed data of the matrix run To review the values in the Matrix Values window Step Action 1 View the Matrix Values window E iz Ponce 6 93 M atrix mtx x Reactions B G af R trem fo2i7o oo000 o0000 05420 1000 foa717 ooo o 32a6 0 5462 10000 o 3515 p 237 0245s oare 10000
174. o damages caused by reagents or solvents not supplied by Applied Biosystems even though recommended by Applied Biosystems This Warranty does not extend to any Instrument or part thereof i that has been the subjected of misuse neglect or accident ii that has been modified or repaired by any party other than Applied Biosystems or iii that has been used in a manner not in accordance with the instructions contained in the Instrument User s Manual This Warranty does not cover the customer installable consumable parts for the Instrument that are listed in the Instrument User s Manual 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 the printed product literature or 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
175. odified module files will be saved with a new file name To edit a module Step Action 1 Select a module from the drop down list in Manual Control 2 Click the page icon This opens the Module Editor Settings dialog box Settings for D AppliedBio 310 Modules Seq POP6 1 mL E md4 Injection Time fo set Syring Pump Time poo sec Injection Voltage ps kv Pre Injection EP foo sec Collection Time 120 min NaOH Wash Time p sec EP Voltage 12 2 ky H20 Wash Time 1 sec Heatplate Temperature 50 0 ac HCI Wash Time sec H20WashTime 2 sec Polymer Fill Time p sec Edit the values you want to change Click the Save a Copy button This saves your new settings under a new file name When prompted give the file a descriptive name Note User created module files are not locked Editable Parameters The parameters that can be edited differ from module to module You may be able to Injection time Injection voltage Run voltage Run time Syringe pump time Run temperature Pre injection electrophoresis time Except for syringe pump time and pre injection electrophoresis time the parameters above can be altered for individual samples through the injection list without permanently modifying the module See Chapter 5 Injection Lists as well as Editing Injection Lists on page 6 17 DNA sequencing or 7 16 fragment analysis The settings
176. of the chemicals in the waste container before you store handle or dispose of chemical waste Handle chemical wastes in a fume hood Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals e g safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e g fume hood For additional safety guidelines consult the MSDS After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations A site preparation and safety guide is a separate document sent to all customers who have purchased an Applied Biosystems instrument Refer to the guide written for your instrument for information on site preparation instrument safety chemical safety and waste profiles Some of the chemicals used with this instrument may be listed as hazardous by their manufacturer When hazards exist warnings are prominently displayed on the labels of all chemicals Chemical manufacturers supply a current MSDS before or with shipments of hazardous chemicals to new customers and with the first shipment of a hazardous chemical after an MSDS update MSDSs provide you with the safety inf
177. on time or voltage Current too high Decomposition of urea in the polymer a Clean the syringe and pump block b Install new polymer Incorrect buffer formulation most likely too concentrated Replace buffer with appropriate 1X running buffer Arcing to conductive surface on the instrument Clean the heat plate electrode thumbscrew and autosampler Ensure that the ambient temperature is between 15 and 30 C and the humidity is below 80 Check for excessive condensation on the instrument Troubleshooting 8 7 Signal Strength and Quality Problems 8 8 Troubleshooting Problems with Signal Strength and Quality Observation Possible Causes Recommended Actions No signal No sample added a Check pipet calibration b Examine the efficiency of the PCR and or cycle sequencing reactions c Remake sample Template not accurately quantitated DNA sequencing Quantitate template Refer to the sequencing kit protocol for appropriate DNA quantities Sample not at bottom of tube Spin sample tube in microcentrifuge Air bubble at bottom of sample tube Spin sample tube in microcentrifuge to remove air bubbles Volume of sample too low Sample volume must be at least 10 uL Capillary misaligned with cathode Align capillary and cathode Note Thecapillary should be adjacent to but not touching the cathode The capillary should p
178. or details No firmware All three steady 2 4 Overview of the 310 Genetic Analyzer Back of the Instrument Diagram Service LEDs 1 Serial connections Service LEDs 2 Reset button On Off switch Power cord receptacle Note For an explanation of the service LEDs see the table below Service LEDs The following table lists the service LEDs in the above figure The LEDs are red lights The pattern of lights indicates the instrument s state LED Purpose 1 INIT Indicates board is functioning properly SPARE Not used TRAY Indicates 48 or 96 well tray On 48 well tray Off 96 well tray Z LIMIT Y LIMIT X LIMIT Lit when the autosampler is at the furthest extent of its travel When the electrode is at the front left of the autosampler all lights should be on 2 FIRMWARE STATUS Lights 2 through 9 indicate the status of the firmware Flashing lights indicate the firmware is loaded All lights off mean the board has a problem Four lights on and four off means you need to reload the firmware XLX When flashing indicates the logic board is functioning properly Overview of the 310 Genetic Analyzer 2 5 Serial Connections Reset Button On Off Switch Power Cord Receptacle The following table lists the serial connections on the back of the instrument Port Purpose CONTROL DATA Conn
179. or instrument waste handling and disposal Read the waste profiles and all applicable MSDSs before handling or disposing of waste IMPORTANT Waste profiles are not a substitute for MSDS information As the generator of potentially hazardous waste it is your responsibility to perform the actions listed below Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial or national regulations Note Radioactive or biohazardous materials may require special handling and disposal limitations may apply Ensure that everyone involved with the operation of the instrument has Received instruction in general safety practices for laboratories Received instruction in specific safety practices for the instrument Read and understood all related MSDSs 7 Ne eh Avoid using this instrument in a manner not specified by Applied Biosystems Although the instrument has been designed to protect the user this protection can be impaired if the instrument is used improperly Operating the computer correctly prevents stress producing effects such as fatigue pain and strain To minimize these effects on your back legs eyes and upper extremities neck shoulder arms wris
180. or two counts per injection for the 1 0 mL syringe If there are This zero counts Indicates that the capillary did not fill and may be clogged more than 15 counts May indicate a polymer leak For more information see Checking for Polymer Leaks on page 3 11 Avoiding Capillary Clogged capillaries cause current problems during electrophoresis Clogs Cause Prevention Proteins Purify your DNA templates Run samples with TSR to avoid drawing protein into the capillary Large molecular weight DNA Purify your DNA templates Run samples with TSR to avoid drawing large molecular weight DNA into the capillary Bacteria Use deionized distilled water Water is the most common source of bacterial contamination Crystallized buffer or polymer Clean the electrode every 2 days Check for crystals at the connection of the glass syringe and gel block Bring polymer to room temperature before use Do not expose the end of a filled capillary to air for more than 30 minutes 3 22 Operating the Instrument Hardware Electrode Overview There are two electrodes in the 310 Genetic Analyzer an anode and a cathode Both electrodes are platinum Electrode Description Anode This electrode is on the left as you face the instrument and is always immersed in the electrophoresis buffer Cathode This electrode is a U shaped wire located abov
181. ormation to narrow the list of possible causes 6 Correct the problem and test the fix Check Our Web Site Often a small amount of time invested in troubleshooting a problem yourself can save you inconvenience Our Web site includes answers to frequently asked questions and allows downloads of software upgrades Applied Biosystems Field Application Specialists Technical Support Specialists Field Service Engineers and Sales Representatives are available to support the 310 Genetic Analyzer fully Please contact the nearest Applied Biosystems office visit us on the Web at http www appliedbiosystems com or call us toll free in the U S see Technical Support on page 1 9 8 2 Troubleshooting Primer Problems Problems with Primer Problems Possible Causes Poor priming Melting temperature is too low due to low GC content and or short resulting in weak primer length signal Secondary structure of the primer particularly at the 3 end Secondary structure of the template in the region of hybridization Adequate signal Secondary hybridization site which results in many extra peaks strength with noisy Impure primer You may see a shadow sequence of N 1 data Troubleshooting 8 3 Polymer Problems 8 4 Troubleshooting Problems with Polymer Observation Possible Causes Recommended Actions Run will not start Not enough polymer for the number of injections indicated in th
182. ormation you need to store handle transport and dispose of the chemicals safely We strongly recommend that you replace the appropriate MSDS in your files each time you receive a new MSDS packaged with a hazardous chemical NUAN CHEMICAL HAZARD Be sure to familiarize yourself with the MSDSs before using reagents or solvents About This Guide 1 5 Ordering MSDSs You can order free additional copies of MSDSs for chemicals manufactured or distributed by Applied Biosystems using the contact information below To order MSDSs Then Over the Internet a Go to our Web site at www appliedbiosystems com techsupp b Click MSDSs Then Enter one of these numbers in the appropriate field on this page Select Click Here then enter the part number or keyword s in the field on this page If you have The MSDS document number or the Document on Demand index number The product part number Keyword s c You can open and download a PDF using Adobe Acrobat Reader of the document by selecting it or you can choose to have the document sent to you by fax or email By automated telephone service Use To Obtain Documents on Demand under Technical Support By telephone in the United States Dial 1 800 327 3002 then press 1 By telephone from Canada To order in Dial 1 800 668 6913 and English Press 1 then 2 then 1 again French Press 2 t
183. ort general settings 4 26 communication retries about 3 52 communication between computer and instrument monitoring 3 52 Communications Diagnostics window 3 52 computer 2 14 Communication Port general settings 4 26 maintenance 3 51 hard drive 3 51 RAM requirements 2 14 System requirements 2 14 computer maintenance 3 51 hard drive maintenance 3 51 consumables DNA sequencing B 4 DNA sequencing part numbers B 4 fragment analysis B 5 fragment analysis part numbers B 6 shared B 2 customer support See technical support 1 9 cycle sequencing 6 5 6 40 1X reactions 6 39 Index 2 D data analysis troubleshooting 8 5 Data Collection software 2 15 installing and configuring 4 17 to 4 28 RAM requirements 2 14 detection 2 20 detection region parts of 2 10 detection system positioning capillary in the detector 3 17 DNA sequencing about 2 18 application kits part numbers B 4 color guide for displaying raw data 2 21 consumables part numbers B 4 cycle sequencing using terminators 6 5 Electrophoresis Running Voltage about 6 18 injection list how to fill out 6 15 to 6 16 matrix standards part numbers B 4 modules 6 19 preparing reactions 6 38 sequencing cycle standards part numbers B 4 sequencing standards 6 35 switching to from fragment analysis appplication 3 50 DNA templates amount of DNA to use 6 36 poor preparation 6 35 potential contaminants 6 35 preparation 6 34 quality 6 35 Documents on Demand 1 12 dRhodamine m
184. osition it over the syringe plunger From the Function menu select Syringe Down Select 50 step intervals and click Execute until the toggle makes contact with the syringe plunger Operating the Instrument Hardware 3 29 Heat Plate Preheating the Heat This procedure is optional Preheating the heat plate can take up to 30 minutes You Plate can prepare the samples while the instrument preheats To preheat the heat plate Step Action 1 Open the Data Collection software if not open already 2 From the Window menu select Manual Control The Manual Control window opens From the Function menu select Temperature Set EL lxi Function 26 Value Range E t Temperature Set 20 0 20 0 to 75 0 C Secure Module zone sj Dj The Value text box becomes active Type the temperature in the Value text box according to the table below Application Temperature C DNA sequencing 50 Fragment analysis 60 Click Execute IMPORTANT The instrument doors must be closed The safety interlock prevents operation when the doors are open Prepare your samples while the instrument is preheating Note There is no active cooling system on the 310 Genetic Analyzer You cannot set the heat plate to a temperature lower than the ambient temperature 3 30 Operating the Instrument Hardware Sample Trays Tray Types One of two trays can be at
185. otometer prior to loading on the 310 Genetic Analyzer To ensure reproducibility of results for all samples prepare the formamide size standard mix using the 12 1 ratio of reagents stated in the procedure below To prepare the formamide sample mix Step Action 1 Prepare the 12 1 formamide size standard PCR sample mix a Prepare the formamide size standard 12 0 uL deionized formamide 0 5 uL size standard such as GeneScan 400HD ROX b Mix in a sample vial 12 0 uL formamide size standard 1 uL PCR sample c Label the vial d Vortex the mixture gently for 3 to 5 seconds e Spin down the mixture f Store the mix at 2 to 6 C until ready to use 2 Cover the tube with a septum and vortex well Setting Up Fragment Analysis Experiments 7 7 Diluting the Formamide Sample Mix Minimum Sample Volume Loading the Samples To prepare the formamide sample mix continued Step Action 3 Heat the mixture for 2 minutes at 95 C and place it on ice until ready to place in the instrument The 12 1 ratio of formamide size standard PCR sample mix is usually an adequate dilution however if the peaks go off scale at this dilution further dilute the mixture To dilute the mixture transfer into a sample tube 1wuLofthe 12 1 formamide size standard PCR sample mix 9QuL of formamide The minimum sample volume per tube is 10 uL If you choose to use 10 uL samples rou
186. ours you will receive an e mail reply to your question from an Applied Biosystems technical expert To Obtain Technical You can obtain technical documents such as Applied Biosystems user documents Documents MSDSs certificates of analysis and other related documents for free 24 hours a day You can obtain documents By telephone Through the Applied Biosystems web site 1 12 About This Guide Ordering Documents by Telephone To order documents by telephone 1 From the U S or Canada dial 1 800 487 6809 or from outside the U S and Canada dial 1 858 712 0317 2 Follow the voice instructions to order documents for delivery by fax Note There is a limit of five documents per fax request Obtaining Documents Through the Web Site To view download or order documents through the Applied Biosystems web site Step Action 1 Go to http www appliedbiosystems com 2 Click SERVICES amp SUPPORT at the top of the page then click Documents on Demand 3 In the search form enter and select search criteria then click Search at the bottom of the page 4 In the results screen do any of the following Click the pdf icon to view a PDF version of the document Right click the pdf icon then select Save Target As to download a copy of the PDF file Select the Fax check box then click Deliver Selected Documents Now to have the document faxed to you Select the Email chec
187. out This Example iost sotnii e Re Ree MER eee bees 7 25 Reviewing the Data 5 0600 ees bee tea ER eb RENE SRS SMES ees oF EE SE 7 25 Making a Matrix File i a E cc eee eee 7 26 Checking the Matrix Quality 0 0 0 eee eee 7 27 Inspecting the Matrix Quality 0 eee eee 7 28 8 Troubleshooting Introduction 2o Let faced SUR Go i aces b Acne EMEN ed PPP Seis 8 1 In Fs Ch pter 4 er ROSE END es Sth eee Rp e OR EAs EUR eS 8 1 How to Troubleshoot Data 0 0 ccc cee m e 8 2 Troubleshooting Data cu eens ian a ea Sa ees RR eee RS 8 2 Check Our Web Site sp senses a ea LRL eae ae ae tant eee es 8 2 Primer Problems ober LE dicks oani SS eE ai nance e get 8 3 Polymer Problems cyan seed a Dobos hese SoS eee RARER RES ER ECT DUE 8 4 Automatic Data Analysis Problems 00 0 e cece ee 8 5 Current Problems 4e ete VER tot ee ES oe hae SE E eS eo ee ee ON 8 6 Signal Strength and Quality Problems 0 0c e eee eee eee eee 8 8 Peak Number and Position Problems 0 0 c cece eee ete nh 8 12 Problems with Peak Quality and Resolution 0 0 cece eee eee eee 8 16 Problems with Poor Amplification 2 0 0 0 0 0 0 eee ee eee eee nee 8 18 Problems with Extra Peaks sister it a Wah Fae DOG GR SN So boas 8 22 A Limited Warranty To What This Warranty Applies 0 0 cece eee eee nee eee eee A 1 To What This Warranty Does Not Apply 00 0 cece eee eee e A 1 Ap
188. ples adding to injection list 5 10 adding to instrument mid run 5 11 adding using injection list 5 9 changing order in injection list 5 7 denaturing and loading DNA sequencing 6 9 to 6 10 fragment analysis 7 7 to 7 8 denaturing the matrix standard samples fragment analysis 7 24 sequencing 6 26 evaluating samples using an agarose gel 8 20 generated matrix file from a sample 6 30 loading fragment analysis 7 8 sequencing 6 10 Index 6 loading on autosampler 3 9 making multiple injections 5 5 minimum sample volume fragment analysis 7 8 sequencing 6 9 septa strips 3 32 SeqScape software 2 15 2 16 RAM requirements 2 14 Sequencing Analysis Software 2 16 RAM requirements 2 14 sequencing cycle standards DNA sequencing part numbers B 4 Sequencing Injection List Defaults 4 24 Sequencing Sample Sheet Defaults 4 22 sequencing standards 6 35 serial connections back of instrument 2 6 service LEDs 2 5 Set Scale raw data 4 8 short term shutdown 3 44 shutting down 3 44 to 3 45 signal strength troubleshooting 8 8 to 8 11 size standard files 4 26 7 19 size standards part numbers B 5 soft reset performing 3 47 software adding to hard disk 3 51 Data Collection installing and configuring 4 17 to 4 28 other software programs 2 16 starting run of the instrument DNA sequencing 6 22 fragment analysis 7 20 status indicators 2 4 Status window 4 11 to 4 13 Std column fragment analysis 7 11 stopping the instrument 3 49 storing capillary 3
189. plied Biosystems Obligation 0 0 0 cee cece eee eee A 1 What is Conveyed by Purchase A 2 B Consumables Introduction zi eder Ne le Sb cee tan a DER D I RE etin Eo RR B 1 In This Appendix eee e eee exter AD e EU e ARE Ae B 1 OverVI6Ws op beeePRRSH ELE PE REM OE VREPE MEI UR EH P RE Eus B 1 Shared Consumables i icis p ERR Rosae RR RR E E B 2 Capillaries vob a eC eidem B 2 Trays s cen eRe BA GUT ete e RD e o desse nt dbi ed Dd Hale B 2 Other Shared Consumables lleeeeeeeeee e B 3 DNA Sequencing Consumables 0 0 0 0 0 eee e B 4 DNA Sequencing Polymer 2 0 00 0 eee ce e B 4 DNA Sequencing Application Kits 0 0 0 0 cee eee eee eee B 4 DNA Sequencing Matrix Standards 0 00 eee cee eee B 4 DNA Sequencing Cycle Sequencing Standards llle esses B 4 DNA Sequencing Consumables Part Numbers 000 000 eee eee eee B 4 Fragment Analysis Consumables llle B 5 Fragment Analysis Polymer 0 0 00 0 cece cee eee eee B 5 Fragment Analysis Application Kits 0 0 0 cece eee ee eee B 5 Fragment Analysis Matrix Standards 0 0 00 cee eee eee B 5 Fragment Analysis Size Standard Kits 0 0 0 cece ee eee B 5 Fragment Analysis Consumables Part Numbers eese eee B 6 Index About This Guide Introduction In This Chapter This chapter covers the following topics Topic See Page About
190. r To reset the injection counter Step Action 1 Open the Instrument window and select Change Capillary The Reset window opens 2 Click OK in the Reset window to set the injection counter to O zero 3 18 Operating the Instrument Hardware Testing the Capillary Window After the Capillary is Installed After the capillary is installed do the following Calibrate the autosampler after installing a capillary see Calibrating the Autosampler on page 3 4 Test the capillary window see Testing the Capillary Window below Do not turn the capillary fitting with the detector door closed Torque on the capillary can break it Release the capillary from the detector if the capillary fitting needs adjustment Run a test to ensure that the capillary window is clean and positioned correctly before running your samples To test the capillary window Step Action 1 If necessary do the following a Turn on the instrument and the computer b Open the Data Collection software c Create an injection list for instructions for creating injection lists see page 6 15 DNA sequencing or page 7 13 Insert a row into the injection list before the first sample See Adding Rows to an Injection List on page 5 6 for instructions if necessary Set the module to Test CCD 4 Color Change the collection time from 5 minutes to 1 minute When the module runs chec
191. r Hom Autosampler To Position Autosampler Up Autosampler Down Buffer Valve Open Buffer Valve Close Electrophoresis On Operating the Instrument Hardware 3 7 Filling the Buffer The following steps describe how to load buffers and wash solutions on the Reservoir autosampler CHEMICAL HAZARD Some chemicals used with this instrument are potentially hazardous Warnings are prominently displayed on the bottle labels of all hazardous chemicals Material Safety Data Sheets MSDSs are provided by the manufacturer and contain information about physical characteristics hazards precautions first aid spill cleanup and disposal procedures The MSDSs can be kept in the Safety Supplement provided with the chemistry guide Please familiarize yourself with the information contained in these documents before attempting to operate the instrument or using hazardous reagents To fill the buffer reservoir Step Action 1 To access the autosampler on an idle instrument press the Tray button The autosampler moves forward allowing access to all sample and buffer positions Tray button Note On some early 310 Genetic Analyzers the Tray button is located on the front panel where the figure shows the status indicators Fill the anode buffer reservoir to the red line with 1X Genetic Analyzer Buffer with EDTA and install it on the gel block
192. r surface of the stripper plate Be sure you Do not flex the stripper plate upwards while cutting Do not cut the electrode too short Thumbscrew Electrode Stripper plate GR0980 Recalibrate the autosampler after trimming the electrode Operating the Instrument Hardware 3 25 Straightening the The electrode may bend if Electrode A screw top tube is used in position 3 of the autosampler The Z position of the autosampler is homed before the X Y positions The autosampler is not properly calibrated Keys are held down when moving the autosampler with keyboard equivalents The electrode is too long the electrode should be flush with the translucent stripper plate To straighten the electrode if the cathode electrode is bent Step Action 1 Remove the electrode from the electrode thumbscrew 2 Lay it on a flat surface 3 Rub a flatblade screwdriver back and forth along the length of the electrode to straighten it GR1027 4 Replace the electrode 5 Recalibrate the autosampler after installing the electrode 3 26 Operating the Instrument Hardware Gel Block Cleaning the Gel When to Clean the Gel Block Block Glean the gel block when Installing the syringe Removing the capillary Changing between a DNA sequencing experiment and a fragment analysis experiment Shutting down the instrument The instrume
193. ration of the sample template Running a sample standard is optional Applied Biosystems sells sequencing standards for dye terminator and dye primer sequencing Contact your sales representative for more information The Cycle Sequencing Standards provide an additional control to help in troubleshooting electrophoresis runs The following standards are available Dye Primer Cycle Sequencing Standard Dye Terminator Cycle Sequencing Standard dRhodamine Terminator Cycle Sequencing Standard BigDye Terminator Cycle Sequencing Standard 9 9 9 9 BigDye Terminator v 3 0 Sequencing Standard Note For part number information see Appendix B Consumables Inadequate template preparation is the most common cause of sequencing problems Always follow recommended procedures to prepare templates The following are characteristics of badly prepared templates Noisy data or peaks under peaks Failed reactions Weak signal Potential contaminants include Proteins RNAor chromosomal DNA Residual salts Residual organic chemicals e g phenol chloroform and ethanol Setting Up DNA Sequencing Experiments 6 35 Amount of DNA to The amount of DNA template used in a sequencing reaction affects the quality of the Use data Too much template makes data appear top heavy with strong peaks at the beginning of the run that fade rapidly Too little template or primer reduces the signal strength and peak height In the worst case the
194. rations Cutting the Why Cut the Capillary Capillary For some applications you may want to reduce the length of the 61 or 47 cm capillary to reduce the run time A capillary cutter is available for this purpose Capillary cutters are small flat squares of a hard ceramic material You may want to practice this technique on an old capillary before cutting new capillaries How to Cut the Capillary To cut a capillary Step Action 1 Scratch the capillary with the capillary cutter using one firm even stroke 2 Break the capillary at the scratch mark on the glass 3 Examine the capillary The cut end must be flat without jagged edges Cleaning the Clean the capillary window with a lint free lab wipe moistened with ethanol Capillary Window CHEMICAL HAZARD Ethanol is a flammable liquid and vapor It may cause eye skin and upper respiratory tract irritation Prolonged or repeated contact may dry skin Exposure may cause central nervous system depression and liver damage Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 3 14 Operating the Instrument Hardware Installing the About Installing the Capillary Capillary The capillary can break at the detector window if twisted Always connect the capillary to the gel block first because the capillary will turn slightly as you connect it there If the other end is not free to move the capillary
195. rds Kit 403047 dR6G dR110 dTAMRA dROX LIZ 310 377 v 3 0 BigDye Matrix Standards Kit 4390421 Part Sequencing standard Number Dye Primer Cycle Sequencing Standard 401920 Dye Terminator Cycle Sequencing Standard 402830 dRhodamine Terminator Cycle Sequencing Standard 4303120 BigDye Terminator Cycle Sequencing Standard 4304154 BigDye Terminator v 3 0 Sequencing Standard 4390303 Use these part numbers below to order sequencing consumables Part Consumable Purpose Number POP 4 Performance Optimized Polymer for electrophoresing DNA 402838 Polymer fragments in the capillary POP 6 Performance Optimized Polymer for electrophoresing DNA 402844 Polymer with 2 vials of TSR fragments in the capillary POP 6 Performance Optimized Polymer for electrophoresing DNA 403076 Polymer with 8 vials of TSR fragments in the capillary POP 6 Performance Optimized Polymer for electrophoresing DNA 402837 Polymer without TSR fragments in the capillary Mineral oil For use with the DNA Thermal 0186 2302 Cycler TC1 and the DNA Thermal Cycler 480 Fragment Analysis Consumables Fragment Analysis Polymer Fragment Analysis Application Kits Fragment Analysis Matrix Standards Two polymers are available for fragment analysis experiments POP 4 Performance Optimized Polymer GeneScan Polymer for SSCP and native runs A number of kits are available for specialized applications of ABI PRISM GeneScan Analysi
196. rdware 3 35 To clean the syringe continued Step Action 2 Remove the ferrule from the syringe a Soak the ferrule in warm not boiling water for as long as it takes to remove crystals if any in the ferrule b Rinse the ferrule with deionized water Clean the glass barrel with warm pure water Dissolve all crystals if there are any Rinse the glass barrel with distilled deionized water Inspect the O ring in the stainless steel hub of the syringe for damage and replace it if necessary IMPORTANT Make sure the O ring does not block the hole in the stainless steel hub Inspect the O ring on the ferrule and replace it if necessary Place the ferrule back onto the syringe When the syringe is clean do one of the following If Then the syringe is to be used insert the plunger into the barrel count slowly to 5 using deionized water as a lubricant the syringe is to be stored a Dry the plunger and barrel b Insert the plunger into the barrel count slowly to 5 C Store as a matched set IMPORTANT Be sure to store the barrel and plunger as a matched set Filling the Syringe CHEMICAL HAZARD POP polymers may cause eye skin and respiratory tract irritation Please read the MSDS for the polymer you are using and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Use for research and development purposes
197. re File Purpose Both Sample sheet The sample sheet associates sample information name and type of analysis with a sample tube position For more information refer to Chapters 6 and 7 Injection list The injection list specifies the order for running samples how many injections are made from each sample and the module and running conditions for each injection For more information refer to Chapters 5 6 and 7 Module The 310 Genetic Analyzer executes the steps in a module to process a sample Run time temperature electrophoresis voltage injection time and injection voltage are some of the run parameters controlled by the module Matrix Matrix files contain information that corrects for spectral overlap Fluorescent dye labels are sold in sets of four or five The emission spectra of the dyes overlap slightly Although an effort is made to select dyes for each set that overlap minimally spectral overlap still occurs to some extent For more information refer to Chapters 6 and 7 Preference Preference files record the default for a number of different selections in the software Sequence analysis only Basecaller The basecaller file contains the algorithms for calling bases Dye set primer Dye set primer files mobility files adjust the data for the mobility of the sequencing primer or terminator in the polymer Fragment analysis only Analysis parameters
198. rength allows shorter run times but reduces resolution For sequencing samples using The standard running voltage is POP 4 Standard 319 volts cm For a 47 cm capillary this translates to 15 kV The current at this voltage is 7 to 9 pA Temperature is 60 C The time required for a 400 base fragment to reach the detector window is approximately 25 minutes with these run conditions Note The required time listed in the above table is an approximation only The times can vary from instrument to instrument The temperature limits of the heat plate are 60 C down to 5 C above room temperature Note There is no active cooling system on the 310 Genetic Analyzer You cannot set the heat plate to a temperature lower than the ambient temperature You can change the running time for special requirements For example you can shorten the data collection time if you only need information about short extension products Typically you set the electrophoresis running time at an interval 1096 higher than the average migration time of the maximum fragment size of interest to ensure sufficient data collection Setting Up Fragment Analysis Experiments 7 17 Select the Modules Analysis Parameters and Size Standard Files Overview Updated Files Modules Reference Table for Modules Fragment analysis on the 310 Genetic Analyzer requires you to have these types of files Module Size standard Anal
199. rmal Cycler trays to 4305051 be used on the ABI PRISM 310 MicroAmp full plate Seals 96 MicroAmp tubes or plate N801 0550 cover Genetic Analyzer Prevents evaporation of samples and 402059 septa strip consumables The capillary and electrode can pass through a slit in the septum Genetic Analyzer Secures septa strips on 0 2 mL tubes 402866 retainer clip 48 well 48 well sample tray Holds a maximum of 48 0 5 mL tubes in a 5572 4 0 mL 6 x 8 format 0 5 mL sample Holds samples on the 48 well tray as well 401957 tubes as for consumables on the autosampler Septa for 0 5 mL Prevents evaporation of samples and 401956 Other Shared These are the consumables for both sequencing and fragment analysis experiments Consumables Consumable Purpose Part Number Hi Di Formamide Denaturant 4311320 ABI PRISM 10X Running buffer for electrophoresis 402824 Genetic Analyzer Buffer with EDTA for use with POP 4 and POP 6 1 0 mL glass syringe Delivers the polymer to the capillary 4304471 Syringe O rings O rings for the glass syringe and ferrule Two 221102 O rings are required Syringe ferrule Ferrule inside the glass syringe 5401 Cathode electrode Provides the negative pole for electrophoresis 5914 Capillary cutter Shortens the length of the capillary 401958 Capillary fitting Holds the capillary in the pump block 5404 Thermal tape Hold the capillary in position on the hea
200. rotrude 0 5 mm past the cathode Capillary bent out of sample tube Align capillary and cathode Recalibrate autosampler Verify whether a bent capillary is the problem by watching The movement of the autosampler tray during run operation Forinjection current and EP current during data collection Autosampler not calibrated correctly Calibrate autosampler in X Y and Z directions IMPORTANT The capillary should almost touch the Z calibration point Sealed sample tube septum that is septum will not open to allow electrode into sample tube You can either Replace septum or Open the septum by pinching it Septum not placed in the sample tube properly Replace septum Problems with Signal Strength and Quality continued Observation Possible Causes Recommended Actions No signal continued Insufficient injection time Increase the electrokinetic injection time or voltage Signal too low Insufficient sample added a Check pipet calibration b Examine the efficiency of the PCR and or cycle sequencing reactions c Remake sample Dust or particulates have collected on the capillary window and or the internal optics Clean window with ethanol and a lab wipe If no improvement contact your service representative to clean the internal optics Capillary not correctly aligned in the window Align capillary in window
201. rt Starting number for automatic file numbering How dyes are represented on screen You can modify the reference files through the Preferences window The Preferences window contains tabs through which you select defaults The files and locations that the Data Collection software will automatically use these default settings unless directed to do otherwise The preference tabs are 9 9 9 9 9 9 9 9 Folder Locations Default File Names Sequence Sample Sheet Defaults Sequence Injection List Defaults GeneScan Sample Sheet Defaults GeneScan Injection List Defaults General Settings Dye Indicators Collection Info To open the Preferences window Step Action 1 Open the Window menu point to Preferences then select the preference file you want to view or modify 2 The Preferences window opens displaying the tab for the preference file you selected You can view different preference files by clicking on the appropriate tab Using the 310 Data Collection Software 4 19 Folder Locations The Folder Locations preferences file tells the injection list where to find the Preferences 4 Sample Sheet folder Module folder Firmware file folder 9 9 9 9 Folder containing Run folders Analysis Settings folder this folder contains basecaller files GeneScan Analysis Parameters folder GeneScan Size Standard folder The name in the button identifies the path to a folder Clicking the button that show
202. rument a Clean the heat plate electrode and autosampler b Ensure that the ambient temperature is between 15 and 30 C and the humidity is below 80 c Check for excessive condensation on the instrument Irregular peak spacing sequencing Wrong mobility file Use correct mobility file Some but not all loci visible on electropherogram fragment analysis Sample DNA is degraded Quantitate DNA and add more template Repeat amplification Wash the sample in an Amicon Centricon 100 column and repeat amplification Note For fragments smaller than 130 bp the Amicon Centricon 30 column is preferable Sample contains PCR inhibitor for example heme compounds EDTA or certain dyes a Quantitate DNA and add minimum necessary volume of PCR product b Repeat amplification Troubleshooting 8 15 Problems with Peak Quality and Resolution 8 16 Troubleshooting Problems with Peak Quality and Resolution Observation Possible Cause Recommended Actions Fuzzy smeared peaks Too much sample DNA Decrease injection time and or voltage Dilute PCR sample before adding to formamide size standard mix Poor resolution Poor capillary performance Replace capillary Capillary clog especially peaks that start normally but abruptly become broad at some point in run See Avoiding Capillary Clogs on page 3 22 Old defective or i
203. s Used for linkage mapping and human identification applications Other software is available from Applied Biosystems to align sequences identify previously unsequenced regions archive data identify patterns of heredity and other data manipulations See your sales representative or visit the Applied Biosystems Web site at http www appliedbiosystems com The Data Collection software collects the raw data from either DNA sequencing or fragment analysis runs The raw data is fluorescence which is detected on the CCD array as the labeled fragments separated in the capillary by electrophoresis and excited by the laser pass the detection window The raw data is analyzed by the analysis software e g Sequencing Analysis software or GeneScan Analysis software and is converted to DNA sequence data or DNA fragment size quantitation data The Data Collection software contains information about the dyes emission fluorescence spectra and processes the raw data as base sequence fragment size or relative concentration for use by one of the analysis applications The Data Collection software is discussed thoroughly in Chapter 6 Setting Up DNA Sequencing Experiments Overview of the 310 Genetic Analyzer 2 15 Software for Data DNA Sequencing Analysis The DNA sequencing analysis software analyzes the raw sequencing data collected by the Data Collection software The software programs available for analyzing the raw data
204. s Software or ABI PRISM GeneMapper Software with the 310 Genetic Analyzer Please consult your sales representative or see the Applied Biosystems Web site at http www appliedbiosystems com 5 FAM JOE NED ROX Part Matrix standard Number Matrix Standard Set DS 02 for filter set E5 4323050 dR6G dR110 dTAMRA dROX LIZ Matrix Standard Set DS 33 for filter set G5 4318159 6 FAM VIC NED PET LIZ Fluorescent Amidite Standards 401546 6 FAM HEX TET ROX TAMRA NED Matrix Standard 402996 VIC Matrix Standard 4313939 Matrix Standard Set DS 30 6 FAM HEX NED ROX use Fluorescent Amidite Standards kit NED Matrix Standard Set DS 32 for filter set F 4312131 Matrix Standard Set DS 34 for filter set C 6 FAM TET HEX TAMRA use Fluorescent Amidite Standards kit Fragment Analysis Applied Biosystems offers several size standard kits for a variety of applications Size Standard Kits Part Kit For Fragments Between Number GeneScan 120 LIZ 15 bp to 120 bp 4324287 GeneScan 350 TAMRA 35 bp to 350 bp 401736 GeneScan 350 ROX 35 bp to 350 bp 401735 GeneScan 400HD ROX 50 bp to 400 bp 402985 GeneScan 500 TAMRA 35 bp to 500 bp 401733 GeneScan 500 ROX 35 bp to 500 bp 401734 GeneScan 520 LIZ 35 bp to 500 bp 4322682 GeneScan 1000 ROX
205. s Temperature 0 0 0 0 eee cette eee 7 17 About Electrophoresis Running Time 7 17 Select the Modules Analysis Parameters and Size Standard Files 7 18 OVerVIeW soe vida lene eee FERE EIER gig Sanne ges Mane ER PESACUES EOS 7 18 Updated FES in anne i E e ae derek oce rt SUA RR ERG 7 18 Modules ue RETE H Cerere nere She SEND PEN E 7 18 Reference Table for Modules lsseeeeee eh 7 18 Size Standard Files eot ERR e exa uet exuere 7 19 Analysis Parameters Files 0 0 ee 7 19 starting the Run i ca ple EE UE RM ERG RURSUS ook ho e RR c Od ORO EO 7 20 Starting the Rum ost PV Meee e EE E a a 7 20 Monitoring the RUD i nux uU eae eae ee AER eee RUE A e en 7 20 Analyzing Data unte prec p pape UR M ER asia Re E ER EUER 7 20 Section Matrix Files for Fragment Analysis 0c cece een ence ween 7 21 In This Section oe n c SCREENS GL ENSE E SUEERRUS ee eg 7 21 About Matrix Files 4 obRALuS BR ARSeRLIRIIMRRL M Pei 7 22 Matrix Files ehe RRUERPMUREL e RUP SUREC Veg e ee ipie ee det erbe 7 22 How Many Matrix Files sisane rn c ee RR Ro eR Sh ae RR CR as 7 22 About Using Matrix Filesi a II 7 22 Preparing Matrix Standards 0 0 0 ect eeeenen eae 7 23 About Matrix Standards 0 eects 7 23 Preparing Matrix Standard Samples 0 0 00 7 23 Denaturing the Matrix Standard Samples 0 0 00 e eee eee eee eee 7 24 Creating the GeneScan Matrix File 2 0 ee eee eee 7 25 Ab
206. s and Virtual filter that distinguishes one from the other Filters 4 5 FAM and 6 FAM HEX and JOE VIC TAMRA and NED PET and ROX 2 20 Overview of the 310 Genetic Analyzer Color Display of The Data Collection software creates a real time display of the light intensities Data collected on the virtual filters The wavelengths of the dyes in the set are blue green yellow red and orange The colors on the real time display do not represent actual wavelengths They represent the color coded relative wavelengths of the four or five dyes in each dye set Each of the sequencing chemistries is associated with one of the dye sets Each chemistry associates the four bases with dyes of different relative wavelengths Therefore the color associated with each base in the Data Collection software changes with the chemistry used to label it Virtual Filters and In DNA sequencing applications Virtual Filter A uses the four colors to represent Associated wavelengths within the dye set used with Taq primer sequencing and the dye set used Applications With Tag terminator sequencing Virtual Filter uses the four colors to represent wavelengths within the dye set used for primer sequencing with T7 primers an application that is no longer common Virtual Filter E represents wavelengths within the dye set used for sequencing with dichlororhodamine labeled terminators or BigDye labeled primers This virtual filter is also used with
207. s harmful if absorbed through the skin and may cause irritation to the eyes skin and respiratory tract It may cause damage to the central nervous system and the male and female reproductive systems and is a possible birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves The protocol uses a sample preparation reagent designated the TSR 7 Xe eh CHEMICAL HAZARD Template suppression reagent may cause eye skin and respiratory tract irritation Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves When working with TSR keep these points in mind Store TSR at2 to 8 C Atroom temperature samples in TSR are stable for a maximum of 48 hours Although not recommended on a routine basis you can keep samples prepared in TSR frozen for several weeks before running on the 310 Genetic Analyzer with no detectable loss in resolution or base calling To prepare the TSR sample mix Step Action 1 Add 25 uL of TSR to each sample pellet 2 Vortex thoroughly and heat for 2 minutes at 95 C 3 If you are using the 96 well tray assembly you can denature samples directly in the tray See Preparing the 96 Well Sample Tray on page 3 33 Chill samples on ice vortex thoroughly then spin briefly in a microcentrifuge Hold samples on ice or keep frozen until ready to load on the
208. s the path to a folder will allow you to change the path One example of why you may need to do this is if the Modules drop down list in the injection list is empty You may need to reset the path to the Modules folder To set the path to a folder Step Action 1 Click the button that shows the current path to the folder 2 Navigate to the location of the correct folder 3 Select the correct folder then click Select Clicking the Search For Folders button will reset all the folders to the default paths in the AppliedBio folder GeneScan Injection List Defaults E Defaults Sample Sheet Folder Module Folder Folder Containing Run Folders Firmware File Folder Analysis Settings Folder Gene Scan Analysis Parameters Gene Scan Size Standard Folder 4 20 Using the 310 Data Collection Software File Names Preferences x General Settings Dye Indicators Collection Info GeneScan Sample Sheet Defaults Sequence Sample Sheet Defaults Identifies the path to a folder D AppliedBioi310 Sample Sheets D AppliedBio 3 10 Modules D AppliedBiov3 10 Runs D AppliedBio 3 10 Firmweare D AppliedBio Shared Analysis Basecaller Params D AppliedBio Shared Anal ysis Sizecaller Params D AppliedBio Shared Anal ysis Sizecaller Size Standards Search For Folders OK Cancel File Names The Data Collection software creates and names certain files automatically Your Preferences pref
209. sage sent to instrument lt Message received from instrument Warning THER Computer or instrument error 4 12 Using the 310 Data Collection Software Electrophoresis The Electrophoresis History window plots the actual values for the electrophoresis History power supply laser power and heat plate temperature over the course of the run The information in the Electrophoresis History window is stored in each sample file Adjusting the Voltage Current Laser and Temperature Scales to view the Electrophoresis History select Electrophoresis History from the Window menu during a run Electrophoresis History Window Scan Number 20 04 kV 15 0 10 0 504 boo 250 2 3 700 Scan number time scale kv Electrophoresis voltage Electrophoresis current Laser power Heat plate temperature The voltage current laser power and temperature scales are adjustable To adjust scale in the Electrophoresis History window Step Action 1 Double click one of the fields in the window or click a field once then go to the Edit menu and select Scale The Set Scale dialog box opens 2 In the fields of the Set Scale dialog box type the scale s maximum and minimum values 3 Click OK Using the 310 Data Collection Software 4 13 Modules Editing Modules Through the Manual Control window you can customize module files to optimize your runs M
210. sc ebbe o cade hits tb 4 4 Data Collection Software Menus 0 0 0 00 ccc eee eh 4 4 Data Collection Functions lsseeeeeeeee n 4 4 Four Key Tools in the Software 4 4 File Typesc eec in oe ete Gn Se e xu EP EC PELA EE 4 5 Where the Data 1s Stored i io sine eeina ena nea RARE See eee eU Rf 4 5 Manual Control erede ERR REIR exe Rex EAA ere 4 6 OVERVIEW l i olde eS ie a a LEER eae PR e ebd i tel 4 6 Manual Control Functions 0 0 0 0 0 4 6 Open the Manual Control Window 00 eee eee ene 4 7 Exec ting F nctonBs us cese en Oe DE NEU ERR NORTE S 4 7 Starting a Module el pex RE AREE REEL EEG RE E 4 7 Raw Data Display sebo ER DERE SER Nee Ree RA Hr eet vete ene 4 8 OVERVIEW x cosilussetrssebEsteBPesbde e IT Raat Re ea Match wae ONEAN 4 8 Raw Data Window 0 0 0 ee een rs 4 8 Resizing Raw Data eos ec len Ee er ag esa Sess Geen 4 8 Color Buttons 5 b gathad ewok e tet iota abes Naveed ods suites 4 9 Why Raw Data and Analyzed Data Differ in Color 0 0 0 0 0 4 9 vii Data Display after Analysis for DNA Sequencing 00 00000 4 9 Virtual Filters for DNA Sequencing 0 0 cee eee ee 4 10 Virtual Filters for Fragment Analysis 0 0 0 0 c cece eee 4 10 Run Status and History i cr oo ido nee eat oh ey Snes PUE Eee esee 4 11 Monitoring Run Status and History 00 0 0 eee ee eee 4 11 Instrument Run Status 0 0 2
211. scent peak of another dye Dye sets are created to minimize spectral overlap but it still occurs to some extent When choosing matrix files in the sample sheet match the matrix file to the samples by dye set and sequencing chemistry For example samples run with a module file for Virtual Filter G5 should be analyzed with a matrix file that was also run with Virtual Filter G5 The dye set used to create the matrix file must be the same as the dye set used to run the sample If you analyze data with a matrix file that does not reflect the conditions of your run the run will appear to fail Reanalyze with the correct matrix file if you have made this error Create a separate matrix file for each run condition that affects the collection of spectra Some examples are Anew dye is used The pH of polymers or buffers has changed Adifferent type of polymer is used Run the matrix standards under the sample run conditions and make a matrix file Use that matrix file to analyze any and all samples run under those conditions The matrix file contains the information necessary for software to correct the overlap of the dyes emission spectra on the virtual filter Once a matrix file has been created it can be used for subsequent runs performed With the same kit or chemistry Onthe same instrument Using the same Modules Set of dyes Polymer After running the matrix standards use their sample files to generate a matr
212. sheet Sheets Each row in the text file should contain the information for one row in the grid These data fields must also be in the same order as the columns of the grid Everything up to the first tab in the text file is imported into the first field in the grid To import files into a sample sheet Step Action 1 Create a new sample sheet 2 From the File menu select Import 3 In the dialog box that appears choose the name of the file containing the text you wish to import 4 Click OK The corresponding text is imported into the sample sheet Exporting Sample Use the Export command to save the contents of a window grid for example from the Sheets sample sheet into a tab delimited text format This format is useful for exporting to most database word processing or spreadsheet programs To export information to tab delimited text format Step Action 1 Create a new sample sheet and fill in the necessary information From the File menu select Export 2 3 Type a file name in the dialog box that appears 4 Click OK This creates a text file that can be imported into another 310 Genetic Analyzer s sample sheets or viewed in Microsoft Excel or Word applications 7 12 Setting Up Fragment Analysis Experiments Preparing the Fragment Analysis Injection List Overview The injection list specifies the order for running samples how many injections are made from each
213. sly created sample sheet If there is already a sample sheet prepared for the kit go to Preparing an Injection List on page 7 13 To create a new fragment analysis sample sheet Step Action 1 In the Data Collection software select New from the File menu 2 Click GeneScan Smpl Sheet 48 Tube or GeneScan Smpl Sheet 96 Tube as appropriate Create new x Sequence GeneScan Sequence Sequence GeneScan GeneScan Injection Injection Smpl Sheet Smpl Sheet Smpl Sheet Smpl Sheet List List 48 Tube 96 Tube 48 Tube 96 Tube 3 The Sample Sheet window appears Eduntitied 1 Loix GeneScan Sample Sheet 4 Dyes v E Sample Hame Collection Name Color sta Sample Info Comments 4 none A2 none mam L L A3 nones i Note Drag the column markers at the top of columns to change their width Setting Up Fragment Analysis Experiments 7 9 To create a new fragment analysis sample sheet continued Step Action 4 Follow steps 5 through 12 to prepare the sample sheet with or without matrix standards The screens below are examples of GeneScan sample sheets GeneScan sample sheet with matrix standard samples Note Include the matrix standard samples if you need to create a matrix file for the dye set IMPORTANT Do not specify a color in the Std column for the matrix sa
214. t Hardware 3 33 To prepare the 96 sample tray continued Step Action 6 Sit the septa strip on top of the sample tubes with each of the 24 septum placed into the opening of a sample tube Gently and evenly press the septa strip down until the strip is seated securely against the tubes Place the retainer clip over the 24 openings on the septa strip and press down until the clip clicks into place over the rim on each side of the sample tray Inserting the To insert the tray into the 310 Genetic Analyzer 96 Well Tray Step Action 1 Remove the tray assembly from the base for insertion into the 310 Genetic Analyzer Attach the adaptor to the bottom of the tray assembly Sample tray Adaptor cutout With the adaptor cutout pointed toward the instrument take the following actions Place the tray assembly on the autosampler platform Press it flat against the rectangular raised guide until the tray is flush with the platform A sensor in the platform recognizes which tray is present the 96 well tray or the 48 well tray so that the tube positions are correctly identified during the run 3 34 Operating the Instrument Hardware Syringes Overview Warranty Information Basic Components Cleaning the Syringe Glass syringes are used with the 310 Genetic Analyzer and serve as the polymer reservoir during the run This describes
215. t Key with the above keys IMPORTANT By holding the keys down rather than tapping them the autosampler will move in larger increments Use caution when holding down the keys or the autosampler will travel farther than intended causing damage to the electrode and the Teflon block of the autosampler How to Calibrate the Autosampler To calibrate the autosampler Step Action 1 Remove the sample tray and Eppendorf tube from the autosampler IMPORTANT The sample tray must be removed before calibrating the autosampler If the sample tray is not removed the electrode will be bent Open the ABI PRISM 310 Data Collection Software From the Instrument menu select Autosampler Calibration The Autosampler Calibration window opens FS Autosampler Calibration x x x v Y z z Position Rear Home c Rear va Left Right Front Use shift key to decrease step size Press Start Button to begin Cancel Click Start and follow the directions that appear on the screen Move the autosampler using the arrow keys in the Autosampler Calibration window or the arrow keys on the computer keyboard Note Hold down the arrow keys to move the autosampler with larger steps This is often useful for z calibration Operating the Instrument Hardware 3 5 To calibrate the autosampler continued Step Action 6 Align the cal
216. t plate 310021 Gel block Controls the flow of polymer and samples 4303520 through the capillary Waste vial Collects waste from the waste valve on the pump 603796 block Waste valve Attaches the waste vial to the pump block and 604076 controls the flow from the waste vial Plastic syringe luer Attaches the plastic syringe to the pump block 604075 valve and controls the flow from the plastic syringe Anode buffer jar Holds anode buffer 5402 4 mL glass buffer vial Holds buffer and water on the autosampler 401955 with cap adapter Consumables B 3 DNA Sequencing Consumables DNA Sequencing Polymer DNA Sequencing Application Kits DNA Sequencing Matrix Standards DNA Sequencing Cycle Sequencing Standards DNA Sequencing Consumables Part Numbers B 4 Consumables Two polymers are available for DNA sequencing POP 6 Performance Optimized Polymer POP 4 Performance Optimized Polymer Applied Biosystems sells Ready Reaction and Core Kits for the various sequencing chemistries Please consult your sales representative or see the Applied Biosystems Web site at http www appliedbiosystems com Kit Description Ready Reaction Ready Reaction kits contain all necessary reagents in stable premixes Core Kit Core Kits separately package each reagent Part Matrix standard Number ABI PRISM dRhodamine Matrix Standa
217. t the processing of your samples The injection list pulls sample names and associated tube positions automatically from a sample sheet and lists them as sequential injections to the capillary Other information pulled from the sample sheet or created by the injection list is displayed according to settings in the injection list preference files You can change the run parameters for each injection and the order of the injections by editing the injection list Injection Lists 5 3 Working with Injection Lists About the Examples The procedures in this section can be used for both DNA sequencing and fragment analysis applications Using the Fill Down The fill down function is a shortcut for filling in the fields in a column for the samples Function that require the same value name module injection specs etc Note This only works with contiguous fields in a column To use the fill down function Step Action 1 In this example the same module will be used for all the samples For the first sample in the injection list that requires the module click the field in the Module column then click the arrow that appears at the right side of the field A drop down list appears ERR Injection Sheet Untitled 1 x Sample Sheet 022701 kksss ADO o mm Pause lgcancel Length to Detector fo cm Operator Inj Tube amp Sample Name Inj Secs Inj kV Run kV Run C Run Time Finish
218. tached to the tray platform A sensor in the platform automatically detects the type of tray Number of Tube Size of Sample Tubes Positions mL 48 0 5 96 0 2 Sample Numbering The example below shows the numbering of the tube positions in the 48 well tray in the 48 Well Tray 4 On Side 2 2 Because of the staggered arrangement of sample tubes in the 48 tube sample tray the numbering of the tube positions across the first row is A1 A3 A5 etc The numbering of the tube positions across the second row is B2 B4 B6 etc Operating the Instrument Hardware 3 31 Sample Numbering The example below shows the numbering of the tube positions in the 96 well tray in the 96 Well Tray COCO COSCO i 000 D 2 C0000 i 000 on 3 000 ui 000 000000 0000 About Septa Strips Septa strips seal 0 2 mL tubes in the 96 well tray They require septa clips for the 96 Well Tray y er Neha ol The septa strip will melt at high temperatures Do not autoclave or reuse the septa strips F NOTA Do not close the lid when denaturing samples using septa strips in the GeneAmp 9600 or 9700 Thermal Cycler The septa strip may adhere to the lid The retainer clip interferes with the insertion guides in the GeneAmp 9600 and 9700 Thermal Cycler Therefore you cannot place
219. taller disk as a backup copy To reinstall your 310 Genetic Analyzer Step Action 1 From the Start menu point to Settings then select Control Panels Double click the Add Remove Programs icon From the list on the Install Uninstall tabbed page select 310 Data Collection Click Add Remove then follow the prompts to uninstall the software Insert the Installer disk into the computer Double click the Setup icon NI OIO A OJIN Follow the prompts to install the software From time to time you may need to add files for new applications that have been released since the software was written User Bulletins may inform you of a new application requiring updated module and dye set primer files If so the files are available on disks from local representatives or through the Applied Biosystems Web site at http www appliedbiosystems com To install modules and dye set primer mobility files Step Action 1 Copy the modules into D AppliedBio 310 Modules 2 Copy the dye set primer files into D AppliedBio Shared Analysis Basecaller Mobility 4 18 Using the 310 Data Collection Software Data Collection Software Preference Files Overview Preference files set About the Preferences Window 9 9 9 9 Where the software looks for files How files are named Default settings for sample sheets and injection lists Computer instrument communications po
220. tandards The color of standards and the colors present in a sample can also be changed on GeneScan sample sheets Note You cannot switch between 4 Dyes and 5 Dyes in the sample sheet if the sample sheet is linked to an injection list 7 16 Setting Up Fragment Analysis Experiments About Electrokinetic Injection Voltage and Time Electrophoresis Running Voltage for Fragment Analysis Electrophoresis Temperature About Electrophoresis Running Time The electrokinetic injection time and voltage can be varied to regulate the amount of DNA brought into the capillary for analysis The objective is to inject just enough signal into the capillary for adequate peak height good signal to noise while minimizing the peak widths There are no specific requirements for the voltage and current during the electrokinetic injection Injections at 200 volts cm are not distinguishable from injections at 25 volts cm however a lower voltage and current are preferable since the timing of the injection is more accurate Typically a voltage of 50 volts cm is used for the electrokinetic injection At this value injection times of 5 to 60 seconds are required to obtain adequate peak height depending on the application Recommended conditions are provided in the modules however you may customize the parameters Note Excessive injection time can cause loss of resolution Resolution is better at lower field strengths Increasing the field st
221. ter s hard drive and if you need to sequence the same group of samples again you can select the previously created sample sheet If there is already a sample sheet prepared for the kit go to Using a Previously Created Sample Sheet on page 6 13 To create a new DNA sequencing sample sheet Step Action 1 In the Data Collection software select New from the File menu 2 Click Sequence Smpl Sheet 48 Tube or Sequence Smpl Sheet 96 Tube choose the type of tray to be used in the autosampler Create new x Sequence GeneScan Sequence Sequence GeneScan GeneScan Injection Injection Smpl Sheet Smpl Sheet Smpl Sheet Smpl Sheet List List 48 Tube 96 Tube 48 Tube 96 Tube Setting Up DNA Sequencing Experiments 6 11 To create a new DNA sequencing sample sheet continued Step Action 3 The Sample Sheet window appears EA untitied 1 u x Sequencing Sample Sheet 4Dyes 7 Sample Hame DyeSet Primer Matrix File Collection Hame none none snone gt lt none gt nones snone lt none gt none snone none none none none none snone none none snone none none none none none snone none none snone gt none none snone none none snone none none snone gt snone gt none snone gt lsnone gt snone gt lsnone gt none
222. that came on your original system or on the 310 Genetic Analyzer software disks Operating the Instrument Hardware 3 51 Monitoring Communication Between the Computer and the Instrument About the Communications Diagnostics Window Accessing the Communications Diagnostics Window The window shows information for all Send and Reply messages since the Data Collection software was last launched These two types of messages will always be updated The communications diagnostics window displays the Number of messages Number of characters Time out period 9 9 9 Number of retries Number of serial errors Large numbers of retries indicate poor cable routing see Cables on page 3 42 broken pins on connectors or bad cables Serial retries indicate broken pins on connectors or bad cables Long duration for replies indicates a computer serial driver problem To open the Communication Diagnostics window select Communication Diagnostics from the Help menu E Communication Diagnostics Communication Parameters Send Message Packet Q Characters o 9 Timeout 5 a Rees gg Serial Errors If there is no communication the number of characters on Send and Reply will show 0 zero or will stop updating 3 52 Operating the Instrument Hardware Using the 310 Data Collection Software Introduction In This Chapter Topics in this chapter include the following
223. the Electrode lllleeeeeeee e 3 26 Gel Block zo einne CP 3 27 Cleaning the Gel Block 2 2 ccc IIIA 3 27 Removing the Gel Block 0 0 a a E AE IIIA 3 28 Installing the Gel Block 0 cee eee 3 29 Filling the G l Blocks os eei ere ES bbe UE RUE E EESACCES eee 3 29 lchriddbuc E EE 3 30 Preheating the Heat Plate ecce eerie eer Reeriee m P pe eS TER e 3 30 sample Irays 4 sores ep RI sh ME RU NERONI E ahaa Ae 3 31 Tray Types en e PESE exce BON ota bes e decedit tese cete ts 3 31 Sample Numbering in the 48 Well Tray 3 31 Sample Numbering in the 96 Well Tray lesse 3 32 About Septa Strips for the 96 Well Tray 00 0 0 een 3 32 Preparing the 96 Well Sample Tray 0 00 0 e cece eee eee 3 33 Inserting the 96 Well Tray 0 ee RII 3 34 SyIIDPeS eot eWeURDINUROLPSURUSSURE Re RENS UE ERU aeni taia ir 3 35 uc rc 3 35 Warranty Information 00 ccc m 3 35 Basic Component zi ee Pas RR UGLEDOHUIS S PER se ee aes E REI 3 35 Cleaning the Syringe 5 pect e pp Med REG Sa SA a eas 3 35 Filling the Syringe cepe EE babes cca peak ow Reb eet 3 36 Installing the SyrBerws osc edu od woh tls ger ue SLURS eB p pUSPPETRUS 3 37 Inspecting the Syringe 0 0 E E O E E E a a a eae 3 37 Removing Syringes eere ae iae E A a Sa a ege d reet reU 3 37 Setting Syringe Max Travel 0 0 keen cee eee 3 37 Homing the Syringe veo hah ae ie oi aad ed eue 3 38 Handling and Storing Syringes
224. the GeneScan Analysis software Virtual Filters E5 G5 D C and F are used only with the GeneScan Analysis software Color Guide for The following are color guides for DNA sequencing raw data displays DNA Sequencing Applications Virtual Filter A Taq Primer Taq Terminator 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 Virtual Filter E dRhodamine BigDye BigDye BigDye Terminator Terminator Primer v 3 0 Color Base Dye Base Dye Base Dye Base Dye Blue G dR110 G dR110 C dR110 G Dye 1 Green A dR6G A dR6G A dR6G A Dye 2 Yellow C dTAMRA T dTAMRA G dTAMRA T Dye 3 Red T dROX C dROX T dROX C Dye 4 The Sequencing Analysis software converts the information collected by the Data Collection software to a color code in which blue represents C green represents A yellow represents G and red represents T When G is printed black replaces yellow because black is easier to read Overview of the 310 Genetic Analyzer 2 21 Applications Color Guide for The following are color guides for fragment analysis raw data displays Fragment Analysis Corresponding Dye Dye set E5 Dye set G5 Dye set D Dye set C Dye set F Color DS 02 DS 33 DS 30 DS 31 DS 34 DS 32 Blue dR110 6 FAM 6 FAM 6 FAM 6 FAM 5 FAM Green dR6G VIC HEX VIC TET JOE Yellow dTAMRA NED NED NED HEX NE
225. the autosampler 7 24 Setting Up Fragment Analysis Experiments Creating the GeneScan Matrix File About This Example n this example the matrix standards run will be used to make the matrix file For information on preparing the standards refer to Preparing Matrix Standard Samples Reviewing the Data Before making the matrix file review the raw data by viewing and verifying that the raw data from the standards is good Viewing the Raw Data To view the raw data using the GeneScan Analysis software Step Action 1 Create a new project a Select New from the File menu b Select the Project icon An untitled Analysis Control window opens c Select Add Sample Files from the Project menu d Find and open the Run Folder for the matrix standards run e Select the four or five sample files representing the blue green yellow red and orange for 5 dye 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 or five 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 Verifying the Raw Data To verify the raw data in the GeneScan Analysis software Step Action 1 Verify that data pe
226. the capillary is 0 5 mm or less and is aligned with electrode end If you adjust the capillary then recalibrate the autosampler 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 11 minutes Too little sample DNA added to reaction Quantitate DNA and use the amount recommended in the protocol 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 increase time at denaturation plateau and so on PCR Master Mix not well mixed before aliquoting Vortex PCR Master Mix thoroughly Primer concentration too low Use the recommended primer concentration Problems with Poor Amplification continued Observation Possible Cause Recommended Actions Faint or no signal from sample DNA and from positive control continued Primers degraded Use new primers Note Preincubation at 95 C for 5 to 10 minutes should inactivate proteases or nucleases Too little free Mg in reaction Check that you added sufficient total Mg given the total dNTP concentration Note Fr
227. the capillary must protrude well beyond the opening at the tip of the capillary fitting To avoid crushing the opening be certain that you see the capillary in the gel block channel before proceeding to the next step Position the capillary directly below the opening to the glass syringe Tighten the capillary fitting finger tight to secure the capillary IMPORTANT Overtightening can cause cracks in the gel block which will result in polymer leakage IMPORTANT The capillary will twist as the fitting is tightened Leave the other end of the capillary free to twist or the capillary will break 3 16 Operating the Instrument Hardware Positioning the Capillary in the Detector The central portion of the capillary window should rest over the laser detector window without putting strain or tension on the capillary The window is fragile be careful not to break it when you fit the capillary into its track in the detector To position the capillary Step Action 1 Open the laser detector door and position the capillary in the vertical track of the detector Capillary ee e E e Ek Detector plate C 2 EL d BE 9 2 Align the colored labelling mark on the capillary with the top edge of the detector plate and laser detector door This will position the capillary window over the laser detector opening
228. the care use and maintenance of the syringes used with the 310 Genetic Analyzer This information applies to the syringes manufactured by the Kloehn company Following the simple care use and maintenance tips in this bulletin will ensure maximum syringe life and satisfactory results with normal use Each syringe is manufactured to exact tolerances and is tested with deionized water Warranty replacement policy states that syringes will be replaced free of charge if a problem occurs within the first 3 months 90 days of use The lifetime of each syringe varies with use and care The elements of the syringe discussed in this user bulletin are shown below Stainless Plunger Ferrule O rings steel hub Teflon tip Glass barrel Plunger button I Il o oz Qj ol os 05 07 08 09 10m in d er eae eae GR1686 When to Clean the Syringe Thoroughly clean syringes Before loading new polymer Whenever the syringe is removed from the gel block Always verify that the syringe is clean before use How to Clean the Syringe To clean the syringe Step Action 1 Remove the plunger by slowly drawing it from the glass barrel count slowly to 5 i e one thousand one one thousand two while keeping the entire syringe submerged in water IMPORTANT Moving the dry plunger quickly can damage it resulting in premature failure or leakage around the plunger Operating the Instrument Ha
229. the retainer clip on the sample tray while you are using either the 9600 or the 9700 to heat denature your samples You can process fewer samples by leaving the unused wells in the sample tray empty The reaction tube eight strip and septa strip can be cut to accommodate the number of samples you want to process 3 32 Operating the Instrument Hardware Preparing the To prepare the 96 sample tray 96 Well Sample Tray Step Action 1 Place the MicroAmp base on a flat surface do not put the base in the instrument 2 Place the MicroAmp tray onto the base so that the well numbered A1 is located at the upper left corner of the tray The cutout in the outside rim of the tray will then be located on the left side of the tray This orients the tray for proper fit into the sample block Genetic analyzer retainer clip 3 Genetic analyzer septa strip x MicroAmp retainer MicroAmp reaction tubes MicroAmp tray see retainer Note For part number information see Appendix B Consumables Place the MicroAmp reaction tubes in the tray Note The wells in the tray are labeled left to right from 1 through 12 and A through H from back to front Place the MicroAmp retainer over the sample tray and tubes The retainer and tray are keyed so that the retainer can fit only one way Pipette the solution into the sample tubes Operating the Instrumen
230. then click OK to return to the Analysis Control window A typical setting is 2000 6 Click Analyze 7 Choose Results from the Windows menu and check each electropherogram by taking the following action Clicking In the 4 of Panels drop down list 1 Dye Samples 1 on the Sample File side of the Results window 8 If Then the matrix is each peak is one color with the other good colors flat under it the other colors are not flat under the poor peaks or the signal is less than 5000 to 7000 RFU and you consistently see a minor peak in one color underneath a major peak of another color Blue Green 7 28 Setting Up Fragment Analysis Experiments To check matrix quality continued Step Action 9 If the matrix is Then good The matrix file is saved to D AppliedBio Shared Analysis Sizecaller Matrix poor Reanalyze the matrix run a Choose a different start point to generate the matrix file b Reapply the sample file and analyze If this does not improve the matrix data run new matrix standards If this does not work call Technical Support Setting Up Fragment Analysis Experiments 7 29 Troubleshooting Introduction In This Chapter Topics in this chapter include the following Topic See Page How to Troubleshoot Data 8 2 Primer Problems 8 3 Polymer Problems 8 4 Autom
231. tine autosampler calibration is necessary see Calibrating the Autosampler on page 3 4 You can use a single sample for more than one analysis Either inject out of one sample tube repeatedly or split the sample into several tubes Load the samples into the autosampler as follows Step Action 1 If using a 96 well tray fit the tray onto the adaptor base IMPORTANT The tube arrangement and order of the samples in the tray and on the sample sheet must be the same Make note of the tube arrangement you use So that you can prepare the sample sheet correctly 2 Seal each tube with a septum and place the tray into the autosampler 7 8 Setting Up Fragment Analysis Experiments Preparing the Fragment Analysis Sample Sheet About Sample The sample sheet associates sample information name and type of analysis with a Sheets sample tube position in the autosampler Fill out the sample sheet in the Data Collection software to name each sample associate it with a position in the autosampler and make some choices about the sample s processing There are two kinds of sample sheets for fragment analysis GeneScan 48 sample tubes GeneScan 96 sample tubes Preparing a Sample The first time you use a group of samples you must create a sample sheet Sheet The sample sheet will be saved in the Sample Sheet folder on your computer If you use the same type of kit or group of samples again select the previou
232. ting the Instrument Hardware To correct leaks at the valve to the buffer reservoir continued Step Action 4 Plug the other gel block openings with plugs provided in the Basic Installation Kit 5 Clean each gel block channel with distilled deionized water removing and replacing plugs to direct the water through each channel in turn 6 Clean the channel to the buffer reservoir thoroughly 7 Reinstall the block on the instrument If leaks continue at the buffer reservoir valve contact your Service Representative Operating the Instrument Hardware 3 13 Capillary About the Capillary A capillary is a glass tube with a very small internal diameter Filled with polymer it carries the sample past the laser and detector to the gel block The capillary has an opaque polyimide coating except in the window area Through the window the laser and detector read samples during electrophoresis Capillaries are fragile especially at the detection window Do not expose the ends of a polymer filled capillary to air for more than 30 minutes This allows the polymer to dry and adversely affects capillary performance Make sure the autosampler is moved back and the capillary immersed in buffer as soon as samples are loaded Be careful when handling the capillary The capillary window for the detector is very fragile If maintained properly one capillary can be used for at least 100 electrophoretic sepa
233. tive Cap misaligned Ensure that the window is in the groove in the front of the laser Troubleshooting 8 11 Peak Number and Position Problems 8 12 Troubleshooting Problems with Peak Number and Position Observation Possible Causes Recommended Actions Extra peaks in additional colors displayed underneath each peak Note This shows up clearly in analyzed data and may not be an obvious problem in the raw data This is because it is common to have extra peaks in other colors displayed in the raw data Too much sample injected into capillary Problem with sample preparation See Problems with Extra Peaks on page 8 22 Decrease injection time or injection voltage Dilute sample Too little sample injected into capillary matrix overcorrects Increase injection time or injection voltage Quantitate DNA in sample Refer to protocol for correct quantity Incorrect matrix chosen Check matrix selection on the injection list If correct you may want to create a new matrix Incorrect run module filter set chosen Verify run module filter set with dyes being run with charts on page 6 19 and page 7 18 Wrong filter set used Rerun the samples using the correct filter set Many small extraneous peaks appearing next toa high intensity peak Background above Minimum Peak Height value Increase value for Minimum Peak Height Reanal
234. to transfer the samples from the tray compatible with the 9700 after denaturation to the 96 well sample tray before placing it on the autosampler One MicroAmp full plate cover One Genetic Analyzer septa strip One Genetic Analyzer retainer clip 48 welltray 48 well sample tray 4 0 mL 0 5 mL sample tubes Septa for 0 5 mL sample tubes 1 5 mL Eppendorf tube Software Required The following software is required for the procedures described in this chapter ABI PRism 310 Data Collection Software v 3 0 or higher One of the following data analysis software applications GeneScan Analysis software v 3 7 or higher GeneMapper software v 1 0 or higher Summary of To perform a fragment analysis experiment with pooled DNA using the 310 Genetic Procedures Analyzer you need to Step Procedure See Page Set up the 310 Genetic Analyzer as described in Chapter 3 Operating the Instrument Hardware Prepare the matrix standards if necessary Amplify and denature if necessary the samples Load the samples Prepare a sample sheet Prepare an injection list Start the run Create the matrix file if necessary O o N OoOO RAION Analyze the data 7 6 Setting Up Fragment Analysis Experiments Denaturing and Loading the Samples About the Sample Preparation Reagents About Formamide and Samples in Formamide Preparing the Formamide Sample Mix The follo
235. top point for data analysis the default peak height threshold and the default size calling method Size Standard file Holds the results of a run performed with fragments of known length The file can be used to analyze other runs performed under the same conditions to determine the size of fragments of unknown length Autoprint Prints your analyzed data automatically Check the box to have autoprint automatically enabled on a newly created injection list General Settings General Settings sets preferences for communication between the computer and the 310 Genetic Analyzer and sets up automated file numbering Preferences Lx Sequence Injection List Defaults GeneScan Sample Sheet Defaults Folder Locations File Names l Sequence Sample Sheet Defaults GeneScan Injection List Defaults General Settings Dye Indicators Collection Info Global Serial Number 1000 Communication Port E OK Cancel You can set the following General Settings preferences Preference Description Global Serial Number Files can be automatically numbered This is the starting point for automated numbering here Set which files to include in automatic numbering through the file names preferences page Communication Port Tell the Data Collection software which communications port on the computer is connected to the 310 Genetic Analyzer Select the communications port e g COM1 COM2 or No Port to wh
236. trode with lab wipes dampened with water 9 Shut down the computer 10 Turn off the instrument 11 Remove the capillary and store it Operating the Instrument Hardware 3 45 Resetting the Genetic Analyzer About Firmware Firmware is software stored and executed in the 310 Genetic Analyzer s battery backed up memory It communicates with the software on the computer enabling the systems to function together Types of Resets There are three types of reset Type of reset Soft reset Restarts the firmware Cold boot Deletes and reloads the firmware Clear memory Deletes the firmware and calibration data and reloads the firmware You must enter the calibration data again manually When to Reset You should reset the 310 Genetic Analyzer when you experience the following situations Reset the If Instrument Itis behaving unpredictably It does not respond to software commands Computer and the instrument they are not communicating correctly E Communication Error The instrument did not reply to the last message Do you want to disable the COM port for this run Note If No Port is selected in the General Settings in Preferences the computer and the 310 Genetic Analyzer cannot communicate Check this setting and select the correct communications port before resetting Troubleshooting Perform the various types of resets in this order to resol
237. ts Cycle Sequencing Using Terminators DNA Sequencing Chemistries The examples in this chapter use BigDye terminator cycle sequencing chemistry for DNA sequencing on the 310 Genetic Analyzer This method for performing enzymatic extension reactions is quick convenient and commonly used The benefits of this method include the following Single tube reactions Less hands on time required than with dye labeled primer chemistry Same protocol for both single and double stranded templates Less starting template needed than with non cycling protocols Easier sequencing of large constructs compared to non cycling protocols gt 9 9 9 9 More reproducible results There are currently five DNA sequencing chemistries Chemistry Protocol Part Number Dye labeled Taq terminator 402078 Dye labeled Taq primer 402113 Dichlororhodamine dRhodamine 403041 dye labeled terminator BigDye labeled primer v 1 and v 2 403057 BigDye labeled terminators v 1 and v 2 4303237 BigDye labeled primer v 3 4390036 BigDye labeled terminators v 3 4390037 dGTP BigDye labeled terminators v 3 4390038 Dye primer labeling matches a specific dye labeled primer to each of the four bases in four different dideoxynucleotide extension reactions The reaction products are pooled and electrophoresed Dye terminator labeling attaches covalently a specific fluorescent dye to each dideoxynucleotide base Extensio
238. ts hands and fingers design your workstation to promote neutral or relaxed working positions This includes working in an environment where heating air conditioning ventilation and lighting are set correctly See the guidelines below MUSCULOSKELETAL AND REPETITIVE MOTION HAZARD These hazards are caused by the following potential risk factors which include but are not limited to repetitive motion awkward posture forceful exertion holding static unhealthy positions contact pressure and other workstation environmental factors Usea seating position that provides the optimum combination of comfort accessibility to the keyboard and freedom from fatigue causing stresses and pressures The bulk of the person s weight should be supported by the buttocks not the thighs Feet should be flat on the floor and the weight of the legs should be supported by the floor not the thighs About This Guide 1 7 1 8 About This Guide Lumbar support should be provided to maintain the proper concave curve of the spine Place the keyboard on a surface that provides The proper height to position the forearms horizontally and upper arms vertically Support for the forearms and hands to avoid muscle fatigue in the upper arms Position the viewing screen to the height that allows normal body and head posture This height depends upon the physical proportions of the user Adjust vision factors to optimize comfort and
239. u Get an BITO cene otk a oaa eed ied ii ehe 6 29 Checking the Matrix Quality 0 0 0 eee eee 6 29 Creating a Matrix froma Sample 0 0 0 eee ce eh 6 30 OVerVIeW c ndi A beta hase eb PLI ones IEIUNII Bild 6 30 Reviewing the Dat i iussus e Rene Peas Sa ee sae ee 6 30 Making a Matrix File 0 ec eee I 6 30 It You Get an BIO teni Sean ead es tage pedes PERS debe de dea CUEURLEE 6 31 Checking Matrix Quality 0 cee eens 6 32 Section Useful Information for Preparing DNA Sequencing Samples 6 33 In hi Secon zv we eae ee ee oe a eR et E Bee E 6 33 About DNA Template Preparation lleleeeeeee e 6 34 Reference Document eclesie RR ee Eee ye Red 6 34 DNA Template Preparation 6 34 PCR Templates hes OU ELUBPEBEPEENAV E CUR VI e erae nA 6 34 About DNA Template Quality lseeeeeeee e 6 35 Using Control DNA isses RR b a ace eee s CE oa geal 6 35 Sequencing Standards 0 0 ccc e 6 35 Poor Template Preparation 0 0 0 0 eee II 6 35 Contamination 2 7 22 eae mere sine ruben ee aad teeta ea aaa hag 6 35 Amount of DNA to Use 2 neben Stes Ge ete pes Soe ead Sea eat 6 36 About Primer Design scese ast pw say es Ak wR sal ee a ES Ea Ba este ese 6 37 PumesDesigne 5 1 84 hvac a Ri oe Aa ea ha RUE I 6 37 About Preparing Sequencing Reactions 2 0 0 0 cece ee cee eee 6 38 Reagent Age and Reaction Storage 6 38 Reaction TUbesy ce crease hee RR ergo rte ere yee eae TE
240. u can set the size standard dye color for both four dye runs and five dye runs This is the default dye color that will automatically be selected as the color for the size standard on a newly created sample sheet Using the 310 Data Collection Software 4 23 Sequencing Injection When you create a new Sequencing injection list and select a sample sheet the List Defaults software automatically fills out the injection list from the sample sheet You can set Preferences how you want the software to fill out the injection list through the injection list preferences Folder Locations File Names Sequence Sample Sheet Defaults GeneScan Iniection List Defaults General Settings Dye Indicators Collection Info Sequence Injection List Defaults GeneScan Sample Sheet Defaults Length to Detector poc em Operator 4DyeModule lt none gt 5DyeModule none Autoanalyze with none For Sequencing injection lists you can set the following preferences Preference Description Length to Detector The distance between the end of the capillary that is in the sample and the capillary window Enter the length that will automatically appear on a newly created injection list Note The length you enter does not affect the configuration of the instrument It is for your record keeping purposes only Operator Enter the name of the instrument operator that will automatically appear on a newly created injection list 4
241. uccessively into contact with the cathode electrode and one end of a glass capillary filled with polymer An anode electrode at the other end of the capillary is immersed in buffer A portion of the sample enters the capillary as current flows from the cathode to the anode This is called electrokinetic injection The end of the capillary near the cathode is then placed in buffer Current is applied again to continue electrophoresis When the nucleotides reach a detector window in the capillary coating a laser excites the fluorescent dye labels Emitted fluorescence from the dyes is collected by a CCD camera The software interprets the result calling the bases from the fluorescence intensity at each data point The software programs available for DNA sequencing are Sequencing Analysis software SeqScape software Fragment analysis experiments attempt to determine the relative quantity of DNA fragments of a different size in a sample Each sample is labeled with one fluorescent dye A dye labeled internal size standard is also placed in the tube Three samples and a size standard can be electrophoresed simultaneously when using a dye set with four colors When using a dye set with five colors four samples and a size standard can be electrophoresed simultaneously The sample tubes are placed in a tray in the instruments autosampler The autosampler brings each sample successively into contact with the cathode electrode and one end of a g
242. ults Preferences eee een 4 24 GeneScan Injection List Defaults Preferences 0 000000 eee eee 4 25 General Settings shes woh Ns bec pL NER ER BRERA Sak as 4 26 Dye Indicators Preferences 2 0 cece cece teen e teen eens 4 27 Collection Info Preferences 4 28 General Software Procedures 4 2 3 uk gm e e oe RE UR ha a te a eae 4 29 Howto Find Biles 45e 00 6 eta eee tan Sn Rate een ven esaet 4 29 Keyboard Shortcuts sese ONLUS e Ep Rea Bi E ERR 4 29 5 Injection Lists Introduction i Lice IMenbC seb Sent b hacky ebu PE eMe Se odd vedi pushed ERES 5 1 In This Ch ptetziicsteetreeertmgr e pREebeESG PETER RR V RU ERG PA Meet eee eae 5 1 About This Chapter see Db eI RUSWEERAWEOM REVO RE IRE ERES ne 5 2 Procedures Included in This Chapter sleleleseeeeeeeeeeeee 5 2 Procedures Not Included in This Chapter 0 00 0 cee eee ee eee 5 2 About the Injection List leeeeeeeeeee I I 5 3 OV EEVICW iecit sae ee beh deberi x cine p eiui tette temet ed 5 3 Working with Injection Lists leeeeeeeeee III II 5 4 About the Examples anom be DP ENEEVU EAE Ee Nb Reb 5 4 Using the Fill Down Function sesleseeeeeeee Ie 5 4 Changing the Module fora Sample 0 0 eee eee eene 5 5 Making Multiple Injections from One Sample Tube 00000020 ee 5 5 Adding Rows to an Injection List 0 0 0 ee ee eae 5 6 Running a Sample More Than Once 0 0
243. ur LAfeadg 9dOdOLELa yuid dog xuo 9 9dOdOLE ypur3 1u Sdodbes qour L e qaledodoreld 9 dOd s1jojeuiuue BAQbig SJO eululo9 eN azIS 491129 ainpow 93 AMIGO 1eungAes 13 q 1 W JOd Ansjw yo Aedes Medes seg iments xperumi 6 20 Setting Up DNA Sequencing E gourzM eueLA agirdodoLeda qourz eLNL2 agivdodoreda ueej oG x uo 7p tdOdOLE ypur3 1ur bespideurd qour L egeriw aaivdodoreda ueej oG x uo 7p tdOdOLE ypur3 1ur bespisrd qour LM LNLZ dardOd0L da v dOd qow es1e ege W 18Sdg 9dOd Ld JO qour LA ASYELW Ag 9IdOd0l Edd qourleriNre3esaaledodra ueejb dog xu 7p 9dOdOIE vrpura q1u L pideuodoabes 10 qour LA e LIN L2 dajedodoreda qour esje eue LW 31esaaiedodla JO qour LA ASYELW Gg 9dOd0lEdd qow e LWLZ 9Sag 9dOd1ad L A yuid og X w I9 9dOdOLE pew WI 9dOdbes 10 qour LA E LN EZ agjedodoreda 9 dOd sJeuid eAqbig penunuoo sjoultd MeN azIS 491129 ainpow Olly AMIGO 19ungAes 10Aq 1euiA od Ansjw yo Aseyjidea Medes seg Setting Up DNA Sequencing Experiments 6 21 Starting the Run Starting the Run Click the Run button in the Injection List window to start the run Note If you did not preheat the instrument as suggested under Preheating the Heat Plate on page 3 30 it can take up to 20 minutes for the instrument to heat to 50 C Monitoring the Run During the run you can monitor the run and your samples Re
244. urs or 48 injections whichever comes first Polymer Every 3 days Water in the water vial and waste vial Every 48 hours or 48 injections whichever comes first Preparing the P Xez21ie CHEMICAL HAZARD POP polymers may cause eye skin and respiratory Polymer and the tract irritation Please read the MSDS for the polymer you are using and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Use for research and Electrophoresis development purposes only Buffer IMPORTANT 10X Genetic Analysis Buffer with EDTA Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves CHEMICAL HAZARD Some chemicals used with this instrument are potentially hazardous Warnings are prominently displayed on the bottle labels of all hazardous chemicals Material Safety Data Sheets MSDSs are provided by the manufacturer and contain information about physical characteristics hazards precautions first aid spill cleanup and disposal procedures Please familiarize yourself with the information contained in these documents before attempting to operate the instrument or using hazardous reagents To prepare the polymer and the electrophoresis buffer Step Action 1 Equilibrate polymer to room temperature then mix thoroughly by inversion 2 Allow the polymer to sit for at least 5 minutes after mixing 3 Dilute 5 mL of ABI PRism
245. ut the Sample Preparation Reagents 0 0 00 cece eee eene 6 9 About TSR and Samples in TSR 1 0 2 ec eee 6 9 Preparing the TSR Sample Mix 0 0 00 0c eee ee eee 6 9 Minimum Sample Volume 0 0 0 nee eee nee 6 9 Preparing a Portion of a Reaction Mixture for Analysis esses 6 10 Loading the Samples orco pre oie oket WU Wane ba sles ESSERE EP ER p 6 10 Preparing the DNA Sequencing Sample Sheet llle ee eee eee 6 11 About Sampl Sheets ee nerie eve oh eed pe ees I qe ru Gee ae 6 11 Preparing the Sample Sheet 22 0 0 0 0 00 eee eee eee eee 6 11 Using a Previously Created Sample Sheet eese 6 13 Importing Sample Sheets 0 0 0 cee eee 6 14 Exporting Sample Sheets 0 0 eee eee 6 14 Preparing the DNA Sequencing Injection List 0 0 0 e eee eee eee ee 6 15 OVELVICW 53 552 ex ee RIEN MR Sati Oe OG coy Denies Shines d ee Pe 6 15 Preparing an Injection List 2 0 0 0 eee eee 6 15 Editing Injection Lists rnn n a E cc III 6 17 About Electrokinetic Injection Voltage and Time 00 0000 6 17 Electrophoresis Running Voltage for DNA Sequencing 00 6 18 Electrophoresis Temperature 0 0 0 0 cece eee ee 6 18 About Electrophoresis Running Time 0 0 0 0 c eee eee eee 6 18 Selecting the Module and Dye Set Primer File 00 0 0 0 00 e eee eee 6 19 OV erVIeW he seca athe a hd aa ar Cerea iei 6 1
246. ve a problem with the 310 with Resets Genetic Analyzer Soft reset Cold boot Clear Memory Call Technical Support if the problem persists after a Clear Memory reset 3 46 Operating the Instrument Hardware Performing a Soft To perform a soft reset Reset Step Action 1 Push the Reset button on the back of the 310 Genetic Analyzer with a pointed object such as a pen Quit the Data Collection software Reopen the Data Collection software Open the Manual Control window select Syringe Home and click Execute c1 A OIN In the Manual Control window select Autosampler Home X Y and click Execute Autosampler Home Z and click Execute Performing a Cold In Manual Control Boot To perform a cold boot using Manual Control Step Action 1 In the Data Collection software open the Manual Control window Select Cold Boot Instrument from the drop down list Click Execute A dialog box appears as the firmware is sent to the instrument Open the Manual Control window select Syringe Home and click Execute o ci n5 OIN In the Manual Control window select Autosampler Home X Y and click Execute Autosampler Home Z and click Execute If Manual Control is not Working To perform a cold boot without using Manual Control Step Action 1 Quit the Data Collection software 2 Shut down the computer 3 Turn off th
247. wing procedures are for preparing the samples using POP 4 protocols which use highly deionized formamide Hi Di formamide as the denaturant CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and may cause irritation to the eyes skin and respiratory tract It may cause damage to the central nervous system and the male and female reproductive systems and is a possible birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves The protocol uses formamide as a sample preparation reagent Fresh formamide must be deionized and aliquotted into smaller volumes for storage Each aliquot should be adequate for about one week s work When working with formamide keep these points in mind Aliquot in small volumes such as 0 5 to 1 0 mL This will prevent multiple freeze thaw cycles which exposes the sample to water which promotes formamide degradation Store aliquots of formamide at 20 C for up to 3 months Formamide stored at 4 C is good for about 1 week Atroom temperature samples in formamide are stable for a maximum of 48 hours Although not recommended on a routine basis you can keep samples prepared in formamide frozen for no more than three days before running on the 310 Genetic Analyzer with no detectable loss in resolution Unlike DNA sequencing samples fragment analysis samples are not typically quantitated on a spectroph
248. x 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 polymer buffer and run conditions as sample injections Problems with Signal Strength and Quality continued Observation Possible Causes Recommended Actions Noisy baseline Old defective or incorrectly made buffer or polymer Replace buffer or polymer Weak or low signals and or an elevated baseline See Signal Strength and Quality Problems on page 8 8 A noisy baseline in raw data could be electrical noise Contact your service representative Dirty detector window Clean the detector window Plugged broken or nonconducting capillary Replace the capillary Salts in sample leading to injection of insufficient sample Remove ions by Dialyzing sample Ethanol precipitation Spin column purification Capillary not refilling Check for leaks Check for bubbles in the syringe c Replace capillary if clogged omv e d Increase capillary refill time Spikes in baseline Precipitate in polymer Allow polymer to equilibrate to room temperature before adding to capillary Old polymer POP 4 or POP 6 Use fresh polymer Low baseline with no color separation Laser problem CCD problem Contact your service representa
249. xample the matrix standards run will be used to make the matrix file for filter set E BigDye terminator v 1 0 and v 2 0 BigDye Primer v 1 0 or dRhodamine terminator chemistries The Sequencing Analysis software is used to analyze the data For information on preparing the standards refer to Preparing Matrix Standards Reviewing the Data Before making the matrix file review the raw data To review the raw data using the Sequencing Analysis software Step Action 1 View the raw data for all matrix sample files while in the Sample Manager window of the Sequencing Analysis software IMPORTANT Do not analyze the sample files 2 In the Raw Data window verify that the data peaks are present in all four of the standards and that no anomalies are present Note The relative peak amplitude should be less than 4000 y axis of raw data window 3 Note the matrix standard files with the best data for making the matrix file Making a Matrix To make a matrix file from matrix standards File Step Action 1 In the Sequencing Analysis software a Go to the Sample drop down list b Select Make Matrix 2 In the Input file and dye options window Enter number of sample files to use 1 4 5 4 Enter number of dyes 4 5 4 Cancel OK a Set the number of sample files to use to 4 b Set the number of dyes to 4 c Click OK Setting Up DNA Sequenc
250. ype a file name b Click Save The file will be saved in D AppliedBio Shared Analysis Basecaller Matrix If You Get an Error If you get an error message and the software will not make a matrix You may have designated the wrong files The signal may be too weak to make a matrix The signal may be too high over 4000 RFU Note If you do not get adequate signal to make a matrix you will have to rerun the standard Setting Up DNA Sequencing Experiments 6 31 When the Matrix Maker has completed computing the new matrix file the following Checking Matrix This example explains how to apply a matrix to a sample and what to look for Quality To check matrix quality Step Action 1 Open the Sample Manager window in the Sequencing Analysis software 2 Click the Add button 3 Select the sample files to be analyzed in the directory dialog box and choose Finish 4 The P and F checkboxes should not be checked Bring the Matrix File Matrix column of the Sample Manager window into view by scrolling with the horizontal scroll bar Choose the correct matrix for each sample to be analyzed Click Start To check the electropherograms double click on each file to open it then choose the Electropherogram view 7 If Then each peak is one color with the other colors flat under it the matrix is good TGAGTATTCTATAGTGTCACCTAAATAGCT TGGCGTAATCAT 90 100 110 tt bly
251. yringe yring Step Action 1 Install the prepared syringe on the gel block with the plunger positioned at the bottom of its travel 2 Move the syringe drive toggle to the right so it is positioned over the syringe plunger Inspecting the To inspect the syringe Syringe peus Step Action 1 Inspect the glass syringe for two O rings one behind the ferrule and one around the ferrule O rings i em 0 moii 1 0 05 0 1 0 15 0 2 0 25 2 Verify that the ferrule is firmly seated in the end of the syringe Removing Syringes To remove the syringe Step Action 1 Open the Data Collection software if it is not currently open 2 Open the Manual Control window go to the Function drop down list and select Home Syringe 3 Click Execute Unscrew the syringe from the gel block Setting Syringe Max Determine the Syringe Max Travel for each size of glass syringe that you use Record Travel the values on the Calibration sticker on the left side door When you install a syringe enter the correct Syringe Max Travel value in the Function drop down list of Manual Control Operating the Instrument Hardware 3 37 Note The syringe must be empty when setting Syringe Max Travel To set Syringe Max Travel Step Action 1 Open the Data Collection software 2 Open the Manual Control window go to the Function drop down list select Syringe Home t
252. ysis parameters Matrix You select the particular files appropriate to your experiment on the sample sheet and injection list The module files are supplied with the Data Collection software Updated files can be obtained from Applied Biosystems Web site http www appliedbiosystems com Applied Biosystems Technical Support or from your local technical applications specialist call your local sales office for more information For new applications a disk containing the files is often included with the reagent kit Size standard analysis parameters are supplied with the GeneScan Analysis software Matrix files are created using GeneScan Analysis software See the ABI PRISM GeneScan Analysis Software User Guide for more information Modules are selected in the Module column in the injection list Select the module based on the polymer glass syringe size dye set and application you are using Use the following table as a guide when selecting a module Chemistry Module ABI PRISM Linkage Mapping Sets GS STR POP4 1 0mL D HD5 MD10 LD20 Mouse mapping markers Custom primers for fragment analysis AFLP Plant Mapping Kits and II GS STR POP4 1 0mL F StockMarks Kits AFLP Microbial Identification Kit AmpF STR product line except Identifiler SNaPshot Multiplex Kit GS STR POP4 1 0mL E5 High throughput primers for fragment GS STR POP4 1 0 mL G5 analysis 5 dye
253. yze data Sample DNA overloaded Extraneous peaks represent nonspecific DNA comigrating with main fragment peak Load less sample and repeat electrophoresis Too much sample injected into capillary Decrease injection time or injection voltage Problems with Peak Number and Position continued Observation Possible Causes Recommended Actions Extra peaks when sample is known to contain DNA from a single source Samples not fully denatured Make sure the samples are heated at 95 C for 5 minutes prior to loading onto autosampler Unoptimized PCR Check efficiency of the PCR See the GeneScan Chemistry Reference for the ABI PRISM 310 Genetic Analyzer for detailed suggestions Renaturation of denatured samples Load samples immediately following denaturation or store on ice until you are ready to load IMPORTANT Do not store samples on ice for more than 2 hours before loading Note Too much DNA also promotes renaturation but before you add less DNA you will need to assess the signal strength and quality Extra peaks are 1 to 4 nt larger or smaller than expected peak fragment analysis PCR artifact Refer to Problems with Poor Amplification on page 8 18 Size standard peaks not recognized when defining size standard fragment analysis Height of a size standard peak less than the Peak Amplitude Threshold for the size standard color in
254. z GS STR POP4 1 mL F md4 5 150 150 60 24 Bogus Matrix A1 one S STR POP4 1 mL F md4 5 15 0 15 0 60 24 Bogus Matrix A3 two bd A5 three gt A7 four 411 six 3 For each additional sample select a module file from the drop down list in the Module column 5 10 Injection Lists Adding Samples to To add samples to the ABI PRISM 310 Genetic Analyzer instrument during a run the Instrument Mid Run Step Action 1 From the Injection List window click the Pause button Open the instrument doors 2 Eject the sample tray from the instrument by pushing the Tray button on the 310 Genetic Analyzer 3 Add new samples to the sample tray Push the tray button again to return the tray to its original position Close the instrument doors 5 The run will resume automatically Injection Lists 5 11 Setting Up DNA Sequencing Experiments Introduction In This Chapter Topics in this chapter include the following Topic See Page Section Setting Up a DNA Sequencing Run 6 3 About Sequencing Analysis 6 4 Performing a DNA Sequencing Experiment 6 7 Denaturing and Loading Samples 6 9 Preparing the DNA Sequencing Sample Sheet 6 11 Preparing the DNA Sequencing Injection List 6 15 Selecting the Module and Dye Set Primer File 6 19 Starting the Run 6 22 Section Matrix Files for DNA Sequencing 6 23 About Matrix Files 6 24
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