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1. 11570TA Figure 11 The Setup a Results Group window 8 To create a New Plate navigate to the Library and from the Manage menu select Plates then Create 9 Assign a descriptive plate name Select the Plate Type Fragment from the drop down menu Figure 12 Select the appropriate capillary length and polymer a ache i 3500 Series Da f Library Maintenance Tools Manege Preferences Help Log Out nag applied 3 biosystem 5 Assign Plale Contents _ __ Number of Wells 96 96 Fat 384 Run inetrument Capillary Length em ti Secondary Analysis Load Plates for Run 11571TA Figure 12 Defining plate properties 12 Promega Corporation 10 Select Assign Plate Contents 11 Assign sample names to wells 12 In the lower left portion of the screen Figure 13 under Assays use the Add from Library option not shown to select the Assay created in Step 5 Click on the Add to Plate button and close the window al Edit 7 Library Maintenance Tools Manage Preferences Help Log Out NewPlate gt OpenPlate gE SavePlate Close Plate i Import Pl Export JO Find Replace E View Plate Grid Report Qy Print Load Plates for Run Preview Run Monitor Run lilly Review Resurs View Sequencing Results View FragmentHiD Results y S ps Mi AN G ed
2. Use of the GenePrint 10 System with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and POP 7 Polymer GenePrint 10 System Application Note The GenePrint 10 System is Not For Medical Diagnostic Use Guidelines for using the GenePrint 10 System with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and POP 7 Polymer Application Note 211 1 Introduction The GenePrint 10 System was developed and optimized for use with Applied Biosystems Genetic Analyzers using a 36cm capillary and POP 4 polymer However amplification products can be detected using other instrument configurations This Application Note provides general guidelines for use of the GenePrint 10 System with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and POP 7 polymer Optimization may be required for individual research needs See the GenePrint 10 System Technical Manual TM392 for additional information 2 General Considerations for Amplification The GenePrint 10 System is optimized for use with GeneAmp PCR System 9700 thermal cyclers that allow programming of Max mode for the ramp speed See the GenePrint 10 System Technical Manual for amplification conditions For thermal cyclers other than the GGneAmp PCR System 9700 we recommend that you replicate as closely as possible the thermal cycling conditions described in the GenePrint 10 System Technical Manual Suboptimal thermal cycling condit
3. Open the 3500 Data Collection Software The Dashboard screen will launch Figure 1 Ensure that the Consumables Information and Maintenance Notifications are acceptable Set the oven temperature to 60 C then select Start Pre Heat Applied Biosystems recommends that you preheat the oven for at least 30 minutes before you start a run if the instrument is cold To set up an Instrument Protocol navigate to the Library select Instrument Protocols and choose Fragment as the Filter Choose the default protocol that is most similar to the one you will be using from the list of protocols and duplicate it Figure 5 Do not choose Create For example if you are using an 24 capillary Applied Biosystems 3500xL Genetic Analyzer with a 50cm array and POP 7 polymer choose FragmentAnalysis50_POP 7xl and select Duplicate Edit the copy of this Instrument Protocol and fill in the drop down menus as appropriate for your configuration Be sure to choose Fragment as the Application Type and select the dye set that you created in Section 4 B The Run Module will be FragmentAnalysis50O_POP7xl Assign the Instrument Protocol a meaningful name so that it is clear when the Instrument Protocol can be used in future runs Leave all run options and Advanced Options as the default settings You may change default injection parameters later if necessary Select Save and Close When you save this protocol with t
4. See lead elle ll a ee ae Notifications Log aS se E sa aS a 2 a EE eae Desig tie 0S ett eS a ob ee a ae Mes aed 2 ees ae ae he el 2 i lee aot i Passed amp Failed Borrowed Not Calibrated Quality Values Condition Status Mesage 4 v Intensity vs Scan Number EAEE m EER Figure 3 Calibration Run Application Note 211 3 If fewer than the recommended number of capillaries pass the spectral calibration run will be repeated automatically up to two more times Upon completion of the spectral calibration check the quality of the spectral calibration in the Capillary Run Data display Figure 4 Choose either Accept or Reject not shown Note Refer to the 3500 Series Data Collection Software User Manual for the criteria recommended by Applied Biosystems when accepting or rejecting a spectral calibration The data collection software will display the results for passing and failed capillaries If the spectral calibration fails see the Troubleshooting section of the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBDO22 Capillary Run Data Q omw LLL LEE EELEE EAEE Run 1 Run 2 T E eee Run 3 SITES a a a na ie em oo fee eo eee e Overall 2 Passed zi Failed A Borrowed Not Calibrated Capillary 1 Run 1 Quality Value 0 988753 Condition 5 685121 Status Passed Message q 0 989 c 5 685 Intensity vs Scan Number 0 400 800 1200 1600 2000 2400 2800 320 2800 2400 2000 16
5. Set window will appear Name the Dye Set e g Promega 4Dye select Matrix Standard for the Chemistry and select F Template for the Dye Set Template Leave all other settings as the default values You may lock this dye set to avoid making unintended changes Select Save and Close 4 Promega Corporation c To perform a spectral calibration with the Promega 4 dye chemistry choose Maintenance from the Menu bar then choose Spectral in the navigation pane Under Calibration Settings choose Matrix Standard as the Chemistry Standard and select the name of the dye set you created in Step 2 b Check the Allow Borrowing checkbox Select other options as shown in Figure 3 d Select Start Run lec X l Dashboard Edit Library Maintenance Tools Manage v Preferences Help Log Out K Maintenance Of Export Spectral Calibration Results E Signature View Spectral Calibration Report 2n Print een o y y SA ir Calibrate Calibration Settings Current Instrument Consumables 7 Polymer Type POP Capillary Length 50cm ddd Number of Wells 96 96 FastTube 384 Chemistry Standard Matrix Standard Ed Partners ptePostion OA OB Dye Set T Sequencing Install Standard V Allow Borrowing Status Ready Fragment Install Standard J HID Install Standard Y Capillary Run Data 7 Planned Maintenance east
6. anew Sizecalling Protocol navigate to the Library Select Sizecalling Protocols then select Create Assign a descriptive protocol name Figure 8 Select the size standard created in Step 3 Leave all other settings as the default settings Select Save then Close KJE Create New Sizecaling Protocolos I ee Setup a Sizecalling Protocol A i Protocol Name GenePrint10_50cmPOP7xL E Locked lilly Anatyze Instrument Protocols Dye Sets Size Standards j E g Basecalling Protocols t Analysis Settings QC Settings QC Protocols Qe Sequencing Analysis Protocols mecsworonas uistese HIS simtge EIS Snag FragmentAnalysis Protocols is Start Point fo Sizing Start Size fo Primer Peak analy top Pine Sng op Sze e S BSB Common Settings Use Smoothing Use Baselining Baseline Window Pts 51 11566TA Figure 8 The Setup a Sizecalling Protocol window 5 To create a new Assay navigate to the Library Select Assays then select Create In the Assay window Figure 9 select the application type Fragment and the appropriate Instrument Protocol and Sizecalling Protocol Assign a descriptive assay name then select Save and Close PB Create 2 Edit FY Duplicate g Delete p Import Export shy E Signature gt View Audit History View E S
7. 0 Series Data Collection So Dashboard Edit 7 a x ll Livny Maintenance Tools Manage Preferences Help Log Out Library Resources __ E Create 4 Edit E Duplicate f Delete i Import i Export gh E Signature View Audit History View E Signature History AB WHS a biosystems E Filter All Search arr Go ciar Manage J Plates Assays FileName Conventions Setup a Results Group Name GenePrintl0 Preview of Results Group Name GenePrint10 lt Plate Name gt lt Assay Name gt Available Attributes Selected Attributes Injection Number a ze Results Group Name IP Name Select Results Group Attributes Delimiters Assay Name Select a delimiter La Move Down F Add between attributes DES Plate Name Enter a custom value as either the Prefix or Suffix optional pe s Select Reinjection Folder Option Store reinjection sample files in a separate Reinjection folder same level as Injection folders Store reinjection sample files with original sample files same level Select Folder Option Default file location D Applied Biosystems 3500 Data lt IR Folder gt GenePrint10 lt Plate Name gt lt Assay Name gt lt Inj Folder gt Custom file location Browse E Include an Instrument Run Name folder Include a Result Group Name folder F Include an Injection folder
8. 00 1200 800 400 Intensity vs Scan Number k Intensity vs Pixel Number 9324TA Figure 4 The Capillary Run Data display 5 Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer The quality of formamide is critical Use Hi Di formamide Freeze formamide in single use aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Promega Corporation 5 A Sample Preparation Before preparing the samples preheat the oven of the capillary electrophoresis instrument to 60 C as described in Section 5 B Step 1 if desired 1 Determine the number of samples to be analyzed and add additional reactions to this number to compensate for pipetting error Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 ILS 600 and Hi Di formamide as follows 0 5ul ILS 600 x samples 9 5ul Hi Di formamide x samples Note The volume of internal lane standard used in the l
9. 57 14 PM 22 Oct 2012 03 56 06 PM 04 Aug 2010 07 09 36 PM 04 Aug 2010 07 09 36 PM 16 Apr 2013 09 40 30 AM STTTFFF TTT TTF EET ETT EEG EETE 11565TA Figure 6 A list of Instrument Protocols with associated dye set names The arrow indicates the Instrument Protocol created in Step 2 8 Promega Corporation 3 To create a new Size Standard for the Sizecalling Protocol navigate to the Library Select Size Standards then select Create Assign the size standard an appropriate name Figure 7 Choose Red for the Dye Color Type the fragment sizes for the size standard 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases Select Add Sizes Save then Close Note Definition and detection of the 600bp fragment is optional Setup a Size Standard Size Standard Promega ILS600 E Locked Description Dye Color Enter sizes in the field below separated by a comma space or return then click the Add Size s gt gt button to add them to the current size standard definition Enter new Size Standard definition e g 11 0 34 2 55 Current Size Standard definition Delete Selected Sizes 11020TA Figure 7 The Setup a Size Standard window Application Note 211 9 Use of the GenePrinf 10 System with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and POP 7 Polymer 4 Tocreate
10. LA File Name Conventions LA Mi N PE Actions Z GP 10 Assay A GenePrint10 filenames A EY 11572TA Figure 13 Assigning plate contents 13 Under File Name Convention use the Add from Library option to select the File Name Convention created in Step 6 Click on the Add to Plate button and close the window 14 Under Results Groups use the Add from Library option to select the Results Group created in Step 7 Click on the Add to Plate button and close the window 15 Highlight the sample wells then select the boxes in the Assays File Name Conventions and Results Groups that pertain to those samples To assign sample types select the Custom Sample Info tab at the bottom right side of the window assign sample types then save the plate 16 Select Link Plate for Run 17 The Load Plate window will appear Select Yes 18 In the Run Information window assign a Run Name Select Start Run Application Note 211 13 5 C Data Analysis This section describes how to obtain information for analysis of GenePrint 10 System data generated using the Applied Biosystems 3500 series of genetic analyzers with GeneMapper software version 4 1 Note that Promega scientists have not performed a validation of GenePrint 10 data with GeneMapper software version 4 1 using a 50cm capillary and POP 7 polymer Laboratory validation is strongly recommended Some
11. ach dye DG4650 Not For Medical Diagnostic Use Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information GenePrint and PowerPlex are registered trademarks of Promega Corporation Applied Biosystems and GeneMapper are registered trademarks of Applied Biosystems GeneAmp is a registered trademark of Roche Molecular Systems Inc Hi Di and POP 7 are trademarks of Applera Corporation POP 4 is a registered trademark of Life Technologies Inc 1 PROMEGA CORPORATION 2800 WOODS HOLLOW ROAD MADISON WI 53711 5399 USA TELEPHONE 608 274 4330 WWW promega com 2012 PROMEGA CORPORATION ALL RIGHTS RESERVED PRICES AND SPECIFICATIONS SUBJECT TO CHANGE WITHOUT PRIOR NOTICE PRINTED IN USA 6 13 22258 3849 PART AN211
12. denature samples do not close the heated lid as closing the lid may melt the plate septa Place the plate in the 3500 series 96 well standard plate base and clamp the plate retainer squarely in place over the plate and base until it clicks twice Place the plate assembly in Position A on the autosampler with the labels facing you 4 B Instrument Preparation l If you have not already preheated the oven to 60 C select Start Pre Heat Figure 1 Applied Biosystems recommends that you preheat the oven for at least 30 minutes before you start a run if the instrument is cold Library Maintenance Tools Manage Y Preferences Help v Log Out H Edit Existing Plate POP7 Polymer 50cm 24 cap Array 192 vio 28 gt L a 304 336 Samples Remaining 6 87 Days Remaining 6 87 Days Remaining 2 Injections Performed 17 Injections Remaining 48 Injections Remaining 48 Injections Remaining Instrument 3500 Instrument View Instrument Sensor Details Set Temperature to a Oven Temperature C 35 3 EP Off Detection Cell Temperature C 47 1 a Ejro lt Consumables Information o Name onInstrument Expiration Date ju T Part Number POP7 inii i 18 Jan 2013 06 4393708 ABC ini k 08 Jun 2013 07 4393927 CBC temaini i 08 Jun 2013 07 4408256 a 50cm 24 cap jecti jinin 10 Jun 2013 04 4404689 Serial M512F1311 11037TA Figure 1 The Dashboard Application Note 211 Use of the Ge
13. gment Analysis Protocols HID Analysis Protocols gt View Audit Histo ry gt View E Signature History co Gen Date Modified RPO R BLP S S ts ssf oltre ts S S oo slo ula wl E 16HS Protocol L FragmentAnalysis36_POP4xL_ FragmentAnalysis36_POP4_G5 FragmentAnalysis36_POP7x _ FragmentAnalysis36_POP7_G5 FragmentAnalysis50_POP6x _ FragmentAnalysisS0_POP6_G5 FragmentAnalysisSO_POP7xI_1 FragmentAnalysis50_POP7xl_ FragmentAnalysis50_POP7xI_Prom gt FragmentAnalysis50_POP7_G5 GenePrint 21 GenePrint 21longerinj GenePrint10_50cm_POP7 Geneprint10_50cm_POP7xL LongFragmentAnalysis50_PO gt LongFragmentAnalysis50_PO MSL50_POP7xI_1 PP16HS FragmentAnalysis50_POP7x PP16HS FragmentAnalysis50_POP PP16HS FragmentAnalysis50_POP Promega_5Dye_50_POP7 Promega_5Dye_50_POP7xI SNaPshot50_POP7xI_E5 SNaPshot50_POP7_E5 TechServ GenePrint10_50cm_POP7 23 Oct 2012 08 28 34 PM 16 Dec 2011 03 55 20 PM 16 Dec 2011 03 55 20 PM 26 Jan 2012 03 11 16 PM 26 Jan 2012 03 11 16 PM 04 Aug 2010 07 09 34 PM 04 Aug 2010 07 09 34 PM 27 Nov 2012 11 18 20 AM 04 Aug 2010 07 09 34 PM 04 Jan 2013 01 36 10 PM 04 Aug 2010 07 09 34 PM 23 Oct 2012 10 03 04 PM 05 Feb 2013 09 33 35 AM 13 Mar 2013 01 59 46 PM 25 Apr 2013 05 37 34 PM 04 Aug 2010 07 09 34 PM 04 Aug 2010 07 09 34 PM 19 Apr 2013 02 07 56 PM 07 Nov 2012 09 49 45 AM 23 Apr 2013 07 52 22 AM 13 Nov 2012 03 38 04 PM 22 Oct 2012 03
14. he Promega 4Dye dye set the Instrument Protocol name will appear in the Library with the Dye Set Name associated with it Figure 6 Application Note 211 7 Use of the GenePrinf 10 System with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and POP 7 Polymer E Create 4 Edit EQ Duplicate fj Delete MR Import P Export hy E Signature Library Maintenance Tools Manage vY Preferences Help Log Out View Audit History View E Signature History praapplied AS Riesystanis Manage Gs E Cex Setup an Instrument Protocol Sequencing Analysis Protocols Fragment Analysis Protocols Dye Sets Size Standards Basecalling Protocols Application Type Sizecalling Protocols QC Protocols Dye Set Promega 4Dye Instrument Protocol Properties MicroSeqID Protocols Protocol Name Geneprintl0_50cm_POP7xL HID Analysis Protocols ee Run Voltage kVolts 19 5 PreRun Time sec 180 Oven Temperature C Run Time sec PreRun Voltage kVolts 15 Injection Voltage kVolts 1 6 Injection Time sec 15 Figure 5 Setup an Instrument Protocol window ICA Cer 3900 Ser 11564TA Dashboard Edit 1 applied pe SIETE Manage Size Standards Basecalling Protocols Sizecalling Protocols QC Protocols Sequencing Analysis Protocols MicroSeqID Protocols Fra
15. ial dilution 12 0ul JOE from initial dilution 12 0ul TMR from initial dilution 12 0ul CXR from initial dilution 12 0ul Note The dilutions described here may need to be optimized in individual laboratories depending on the sensitivity of each Applied Biosystems 3500 or 3500xL Genetic Analyzer The optimal dilution may differ for each dye fragment Promega Corporation For the Applied Biosystems 3500 Genetic Analyzer 8 capillaries only wells A1 to H1 of the 96 well plate are used for spectral calibration Load 25ul of the fragment mix prepared in Step 3 into each of the eight wells After placing the septa on the plate briefly centrifuge the plate to remove bubbles Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Dry the bottom of the plate to remove any liquid For the Applied Biosystems 3500xL Genetic Analyzer 24 capillaries only wells Al to H3 of the 96 well plate are used for spectral calibration Load 25ul of the fragment mix prepared in Step 3 into each of the 24 wells After placing the septa on the plate briefly centrifuge the plate to remove bubbles Denature samples at 95 C for 3 minutes then imme diately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Dry the bottom of the plate to remove any liquid Note If using a thermal cycler to
16. ignature History applied systems Clear e Is Signed 27 Nov 2012 11 28 15 AM No 04 Jan 2013 01 40 25 PM No 26 Jan 2012 03 11 22PM No 19 Apr 2013 02 0957 PM No 04 Aug 201007 0938 PM No 23 Oct 2012 10 0344PM No 22 Oct 2012 04 2229PM No 23 Apr 2013 07 5226AM No 23 Oct 2012 08 2927 PM No 04 Aug 2010 07 09 38 PM No 04 Aug 2010 07 09 38 PM No 04 Aug 201007 09 38 PM No 07 Nov 2012 09 55 39 AM No 04 Aug 2010 07 09 38 PM No 04 Aug 2010 07 09 38 PM No 04 Aug 201007 09 38 PM No 26 Jan 2012 03 11 22PM No 04 Aug 2010 07 09 38 PM No 16 Apr 2013 10 11 16 AM No File Name Conventions Results Group Setup an Assay Assay Name GenePrintl0_50cm_POP7xL rote pe Protocols Do you wish to assign multiple instrument protocols to this assay No Yes Instrument Protocol Sizecalling Protocol POOH GD HOGHH O GHOGGHO O 11567TA Figure 9 The Setup an Assay window 10 Promega Corporation 6 To create a new File Name Convention navigate to the Library Select File Name Conventions then select Create Figure 10 Select the File Name Attributes according to laboratory practices and save with a descriptive name Under Select File Location choose Default File Location or choose Custom File Location and navigate to your custom location Select Save then Close E 3500 Data Collection Software Dashboa
17. ions can result in imbalanced profiles 3 Materials to Be Supplied by the User e GenePrint 10 System Cat B9510 e PowerPlex Matrix Standards 3100 3130 Cat DG4650 Use of the GenePrinf 10 System with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and POP 7 Polymer 4 Spectral Calibration Using the PowerPlex Matrix Standards 3100 3130 The PowerPlex Matrix Standards 3100 3130 Cat DG4650 contains matrix fragments labeled with four fluorescent dyes fluorescein JOE TMR and CXR Once generated the spectral calibration file is applied during data collection to calculate and compensate for spectral overlap between different fluorescent dye colors These matrix standards are compatible with the Applied Biosystems 3500 and 3500xL Genetic Analyzers For more information see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBDO22 or Applied Biosystems 3500 3500xL Genetic Analyzer User Guide The quality of formamide is critical Use Hi Di formamide Freeze formamide in single use aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal O Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning labe
18. l and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 4 A Matrix Sample Preparation There may be instrument to instrument variation in the sensitivity of detection The dilutions described here may need to be optimized in individual laboratories depending on the sensitivity of each Applied Biosystems 3500 or 3500xL Genetic Analyzer l Thaw the matrix standards Mix each matrix standard by vortexing for 5 10 seconds prior to use Do not centrifuge the matrix standards as this may cause the DNA to be concentrated at the bottom of the tube Note While the matrix dyes thaw preheat the oven of the capillary electrophoresis instrument to 60 C as described in Section 4 B Step 1 Initial dilution of concentrated fragments Before combining the matrix standards dilute the individual matrix standards 1 10 in Nuclease Free Water which is supplied with the PowerPlex Matrix Standards 3100 3130 as described below Vortex for 5 10 seconds to mix Return the undiluted matrix standards to the freezer immediately after use FL JOE TMR CXR Matrix Standard 5ul Sul Sul Sul Nuclease Free Water 45ul 45ul 45ul 45ul Fragment mix using 1 10 dilutions of matrix standards After the initial dilution in Step 2 combine the 1 10 dilutions of the matrix standards as directed below Vortex for 5 10 seconds to mix Component Volume Hi Di formamide 652ul FL from init
19. nePrinf 10 System with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and POP 7 Polymer 2 To perform a spectral calibration with the Promega 4 dye chemistry for the first time create a new dye set a To create the new dye set navigate to the Library highlight Dye Sets and select Create Figure 2 E Dashboard Edit gt Library Maintenance Tools Manage Preferences Help Log Out Library Resources BB Create A Edit E Duplicate gfi Delete i Import 2 Export i E Signature View Audit History View E Signature History m so Ger Calibration Date Capillary Array Serial Number Setup a Dye Set Size Standards Dye Set Name Promega 4Dye Locked Chemistry Matrix Standard me Dye Set Template F Template g c Arrange Dyes Dye Selection Reduced Selection Calibration Peak Order 4 Basecalling Protocols Sizecalling Protocols fc Protocols Sequencing Analysis Protocols MicroSeqID Protocols Fragment Analysis Protocols HID Analys is Protocols aa SS Parameters The parameters will be used for instruments configured with 36cm capillary array and polymer POP4 Matrix Condition Number Upper Limit 8 5 Locate Start Point After Scan 750 Before Scan 5000 Limit Scans To 2500 Sensitivity 0 4 Minimum Quality Score 0 95 9322TA Figure 2 The Create New Dye Set window b The Create New Dye
20. oading cocktail can be increased or decreased to adjust the intensity of the size standard peaks Vortex for 10 15 seconds to mix Add 10ul of loading cocktail to each well Add 1ul of amplified sample or 1ul of GenePrint 10 Allelic Ladder Mix to each well Cover wells with a plate septa Note Instrument detection limits vary therefore injection time or injection voltage may need to be increased or decreased to optimize peak heights To modify the injection time or injection voltage in the run module select Instrument Protocol from the Library menu in the data collection software Centrifuge plate briefly to remove air bubbles from the wells Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Note If using a thermal cycler to denature samples do not close the heated lid as closing the lid may melt the plate septa 5 B Instrument Preparation Refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide for the instrument maintenance schedule and instructions to install the capillary array buffers and polymer pouch and perform a spatial calibration This Application Note provides general guidelines for use of the GenePrint 10 System with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and POP 7 polymer Some optimization may be required for individual research needs l
21. optimization may be required To analyze GenePrint 10 fsa files collected on the Applied Biosystems 3500 series of genetic analyzers download the panels and bins text files and GenePrint 10 GeneMapper software version 4 0 analysis method at www promega com gene printsupport Make the following selections GenePrint 10 and GM Files Enter your contact information make sure the permission box in the lower left corner is checked and select Submit You will receive an e mail with a link to download the requested files For instructions about basic data analysis contact Promega by e mail at genetic promega com Representative data are shown in Figure 14 DO3_2800M 4_10ng_12 fs GenePrit_10 v10 B B 20 120 160 200 240 280 320 360 g iii 0O03_2800M 1_10ng_12 fsa D55818 AE iv 11573TA Figure 14 Representative data for the GenePrint 10 System Ten nanograms of 2800M Control DNA was amplified using the GenePrint 10 System Amplification products were mixed with Internal Lane Standard 600 and injected for 15 seconds at 1 6kV on an Applied Biosystems 3500xL Genetic Analyzer with a 50cm array using POP7 polymer The resulting fsa data file was analyzed using GeneMapper software version 4 1 an imported HID analysis method file and GenePrint 10 panels and bins text files Ordering Information Product Size OF GenePrint 10 System 50 reactions B9510 PowerPlex Matrix Standards 3100 3130 25ul e
22. rd Edit Y Library Maintenance Tools Manage Y Preferences Help Log Out Library Resources ED Create Edit EY Duplicate g Delete Ae Import i3 Export soy E Signature View Audit History View E Signature History Filter All Setup a File Name Convention Name Promega File Name Locked Select File Name Attributes Preview of File Name lt Sample Name gt _ lt Well Position gt Available Attributes Selected Attributes Unique Time Stamp Integer Sample Name User Defined Field 1 Underscore _ User Defined Field 2 Well Position User Defined Field 3 User Defined Field 4 User Defined Field 5 User Name v Delimiters Select a delimiter Underscore _ Add between attributes Add a custom value to available attributes optional Custom Text 1 Custom Text 2 Custom Text 3 E i Select File Location i a Default File Location D Applied Biosystems 3500 Data N wa A Custom File Location cose 11024TA Figure 10 The Create New File Name Convention window Application Note 211 11 Use of the GenePrinf 10 System with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and POP 7 Polymer To create a new Results Group navigate to the Library Select Results Group then select Create Figure 11 Select Results Group Attributes according to laboratory practices Save with a descriptive name and select Close 350

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