Use of the MSI Analysis System with the Applied
Contents
1. Promega Use of the MSI Analysis System with the Applied Biosystems 3500 Genetic Analyzer and POP 7 Polymer MSI Analysis System Application Note Promega Corporation Notes The MSI Analysis System is For Research Use Only Not For Use In Diagnostic Procedures Guidelines for detecting microsatellite instability using the MSI Analysis System Applied Biosystems 3500 Genetic Analyzer and POP 7 Polymer 1 Introduction The MSI Analysis System was developed and optimized for use with Applied Biosystems Genetic Analyzers using POP 4 polymer and a 36cm capillary array However ampli fication products can be detected using other instrument configurations This Application Note provides general guidelines for use of the MSI Analysis System with the Applied Biosystems 3500 and 3500xL Genetic Analyzers and POP 7 polymer Optimization may be required for individual research needs 2 General Considerations for Amplification The MSI Analysis System is optimized to amplify 1 2ng of genomic DNA in a 10ul reaction volume using the GeneAmp PCR System 9600 and 9700 thermal cyclers See the MSI Analysis System Version 1 2 Technical Manual 1M255 for amplification conditions For thermal cyclers other than the GeneAmp PCR System 9600 and 9700 we recom mend that you replicate as closely as possible the thermal cycling conditions described in the MSI Analysis System Version 1 2 Technical Manual 1M255 Subo2. Component Volume Hi Di formamide 652ul FL from initial 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 On the Applied Biosystems 3500xL Genetic Analyzer 24 capillaries only wells A1 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 On the Applied Biosystems 3500 Genetic Analyzer 8 capillaries only wells Al 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 8 wells After placing the septa on the plate briefly centrifuge the plate to remove bubbles 5 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 Note If using a thermal cycler to denature samples do not close the heated lid as this may melt the plate septa 6 Place the plate in the 3500 series 96 well standard plate base and clamp the plate retainer squarely in place over
3. Size Standard PPlex_ILS600 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 10902TA Figure 6 The Create New Size Standard window MSI Analysis System 4 To create a new Sizecalling Protocol navigate to the Library Select Sizecalling Protocols then select Create Assign a descriptive protocol name Figure 7 Select the size standard created in Step 3 Leave all other settings as default Select Save then Close E 3500 Data Collection Software Dashboard Edit Library Resources i mE e e Setup a Sizecalling Protocol Plates Assays File Name Conventions Protocol Name MSLILS600 Locked Results Group Instrument Protocols Dye Sets Size Standards Basecalling Protocols QC Protocols Sequencing Analysis Protocols MicroSeq D Protocols Fragment Analysis Protocols Library Maintenance Tools Manage v Preferences Help Log Out HID Analysis Protocols FA 7 bx A NT WwW Description Size Standard Promega_IL S600 v SizeCaller v1 1 0 v Sizecaller Analysis Settings Analysis Start Point 0 Analysis Stop Point 1000000 Analy
4. and select the dye set that you created in Section 4 B The Run Module will be FragmentAnaysis50_POP7 Assign the Instrument Protocol a meaningful name so that it will be clear what the Instrument Protocol can be used for in future runs Leave all run options and Advanced Options as the default values It is possible to change default injection parameters later if necessary later Select Save and Close When you save this protocol with the Promega 4Dye dye set the Instrument Protocol name will appear in the Library with the Dye Set Name associated with it 3500 Data Collection Software ECRI Dashboard Edit Library Maintenance Tools Manage Y Preferences Help Log Out Library Resources F Create Edit w Duplicate ff Delete l Import i Export A t View Audit History X Manage Plates Assays Instrument Protocol Name Type Dye Set Name Description Date Modified Is Signed File Name Conventions ES FragmentAnalysis50_POP6x_1 Fragment 65 15 May 2009 05 17 01PM__ No 2 FragmentAnalysis50_POP6_1 Fragment 65 15 May 2009 05 17 0LPM No Results Group ri m Analyze 4 5 Setup an Instrument Protocol 6 Dye Sets 7 Size Standards e 4 Basecalling Protocols 7 Sizecalling Protocols ETE Application Type Capillary Length cm Polymer POP v QC Protocols Sequencing Analysis Protocols Dye Set MicroSegqiD Protocols PAEA AEEA Instrument Protocol Properties HID Analysis Protocols Run Module Giaai
5. should not interfere with data interpretation Other nonspecific artifacts may occur depending on the detection conditions See the troubleshooting section of the MSI Analysis System Version 1 2 Technical Manual 1M255 for more information 16 File Edit View Plots Tools Alleles Help amp A Plot Setting Traditional Genotype Plot Pores 4r DED Bee D e Mite ee A Sample Name sos SQ SSPK MIX OMR cCGQ pos pr 10 maart A if A A B Mark Sample for Deletion Mark Sample for Deletion 25 65 105 145 185 225 wr aww a 2 a a pos pr 10 maart A iB A A C Mark Sample For Deletion 25 65 105 145 185 225 To analyze fsa files collected on the Applied Biosystems 3500 series of genetic analyzers using GeneMapper software version 4 1 download the panels and bins text files at www promega com resources tools msi panels and bins for genemapper software Install the panels and bins text files as described in the MSI Analysis System Version 1 2 Technical Manual 1M255 For data import and data processing follow the instructions provided in the MSI Analysis System Version 1 2 Technical Manual TM255 To analyze fsa files collected on the Applied Biosystems 3500 series of genetic analyzers using GeneMapper JD or GeneMapper D X software contact Promega Technical Services by e mail at genetic promega com to obtain the appropriate panels and bins text 10909TA
6. 04 4404689 Serial M512F1311 50cm 24 cap Priority Notification Date 11037TA Figure 1 The Dashboard MSI Analysis System 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 b The Create New Dye Set window will appear Name the Dye Set e g Promega 4Dye select Matrix Standard for the Chem istry 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 Dashboard Edit Library Maintenance Tools Manage Preferences Help Log Out Library Resources MB create A Edit FY Duplicate fi Delete i Import EA Export i E Signature View Audit History View E Signature History Filter All Search Go Clear aie Plates Assays Faq Dye Set Chemistry Standard Calibration Date Capillary Array Serial Number Is Signed File Mame Conventions Results Grou eae Setup a Dye Set Analyze Instrument Protocols Size Standards Dye Set Name Promega 4Dye Locked Basecalling Protocols Sizecalling Protocols Mc Protocols Sequencing Analysis Protocols MicroSeqID Protocols Fragment Analysis Protocols HID Analy
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 left 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 Promega 15 MSI Analysis System 5 C Data Analysis This section describes how to obtain information for the analysis of MSI Analysis System data generated using the Applied Biosystems 3500 series of genetic analyzers with GeneMapper software version 4 1 Note that Promega scientists have not verified the performance of GeneMapper software version 4 1 with MSI Analysis System data generated using a 50cm capillary and POP 7 polymer Some optimization may be required l For detailed data analysis instructions refer to the Applied Biosystems GeneMapper 4 1 User Guide 5 D Known Artifacts We frequently observe an artifact at 87 90bp in the JOE channel when using POP 7 polymer with the Applied Biosystems 3500 and 3500xL Genetic Analyzers Figure 13 and Applied Biosystems 3100 and 3130 Genetic Analyzers However the size of this artifact is considerably different than the sizes of the repeat markers and as a result
8. Figure 13 An example of an artifact observed when analyzing MSI Analysis System data with an Applied Biosystems 3500 Genetic Analyzer and POP 7 polymer MSI Analysis System a ae Ordering Information Product Size Cat MSI Analysis System Version 1 2 100 reactions MD1641 PowerPlex Matrix Standards 3100 3130 25ul each dye DG4650 For Research Use Only Not for Use in Diagnostic Procedures Not For Medical Diagnostic Use PowerPlex is a registered trademark of Promega Corporation Applied Biosystems and GeneMapper are registered trademarks of Applied Biosystems GeneAmp is a registered trademark of Applera Corporation Hi Di and POP 7 are trademarks of Applera Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information K O PROMEGA CORPORATION 2800 WOODS HOLLOW ROAD MADISON WI 53711 5399 USA TELEPHONE 608 274 4330 Promega www promega comM 2013 PROMEGA CORPORATION ALL RIGHTS RESERVED PRICES AND SPECIFICATIONS SUBJECT TO CHANGE WITHOUT PRIOR NOTICE PRINTED IN USA 8 13 PART AN192
9. The Assay window 10901TA 1 MSI Analysis System EE 6 To create a new File Name Convention navigate to the Library Select File Name Conventions then select Create Figure 9 g ry g Select the File Name Attributes according to laboratory practices and save with a descriptive name Under Select File Location g ry p p choose Default File Location or choose Custom File Location and navigate to your custom location Select Save then Close E 3500 Data Collection Software Dashboard Edit Library Maintenance Tools Manage Preferences Help Log Out Library Resources PB Create 2 Edit E Duplicate g Delete i Import Export i E Signature View Audit History View E Signature History ilter A Go Clea Filter All Go Clear 1 Setup a File Name Convention Results Group m Analyze Name Promega File Name Locked Instrument Protocols Select File Name Attributes 0ye Sels Preview of File Name lt Sample Name gt _ lt Well Position gt Size Standards Available Attributes Selected Attributes Basecalling Protocols Unique Time Stamp Integer Sample Name Sizecaling Protocols User Defined Field 1 Underscore _ QC Protocols User Defined Field 2 Well Position User Defined Field 3 aaa aed User Defined Field 4 MicroSegiD Protocols User Defined Field 5 Fragment Analysis Protocols User Name v Delimiters Select
10. a delimiter Underscore _ Add between attributes Add a custom value to available attributes optional Custom Text 1 Custom Text 2 Custom Text 3 Select File Location Default File Location D Applied Biosystems 3500 Data Custom File Location cose 11024TA Figure 9 The Create New File Name Convention window 12 7 To create a new Results Group navigate to the Library Select Results Group then select Create Figure 10 Select Results Group Attributes according to laboratory practices Save with a descriptive name and select Close f Dashboard Edit Library Maintenance Tools Manage Preferences Help Log Out E j Library Resources X Manage Plates Setup a Results Group Assays File Name Conventions Name MSL Results E Locked Color Select Results Group Attributes Preview of Results Group Name MSI Results lt Plate Name gt lt Injection Number gt Available Attributes Selected Attributes Assay Name Add gt gt Results Group Name IP Name ss JA To eono on lt lt Remove Plate Name v Delimiters Dash Move Up Injection Number Select a deiir Add between attributes Add gt gt Move Down Enter a custom value as either the Prefix or Suffix optional pei sie Tn Select Reinjection Folder Option Store reinjection sample files in a separate Reinjection folder same leve
11. d inhalation and contact with skin Read the warning label and take the necessary 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 Sul 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
12. eat the oven of the capillary electrophoresis instrument to 60 C as described in Section 5 B Step 1 if desired l 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 loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks Vortex for 10 15 seconds to mix Pipet 10ul of loading cocktail into each well Add 1ul of amplified sample Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time or injection voltage may need to be increased or decreased 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 this may melt the plate septa 5 B Instrument Preparation Refer to the Applied Biosystems 3500 3500xL Genetic A
13. l as Injection folders Store reinjection sample files with original sample files same level Select Folder Option Default file location DAApplied Biosystems 3500 Data lt IR Folder gt MSI Results lt Plate Name gt lt Injection Number gt lt Inj Folder gt a Custom file location V Include an Instrument Run Name folder Include a Result Group Name folder V Include an Injection folder Figure 10 The Create New Results Group window 10906TA 13 MSI Analysis System EEE 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 11 Select the appropriate capillary length and polymer 10 Select Assign Plate Contents Dashboard Edit Library Maintenance Tools Manage Preferences Help Y Log Out Plate Name E Start Run Plate Details 0 Define Plate Properties Name Msi 07 03 12 Owner Assign Plate Contents Number of Wells 96 96 FastTube 384 Barcode LA Run Instrument Plate Type a Load Plates for Run f Capillary Length cm Description Preview Run Polymer PoP X Monitor Run m inai gt Secondary Analysis Perform Auto Analysis ees View Sequencing Results lt FE View Fragment HID Results z Figure 11 Defining plate properties 11 As
14. meniioanaeeh Sel 7 envoia Protocol Name MSI50cmPOP7 D Ze escription d eo l Oven Temperature C 60 Run Voltage kVolts 19 5 PreRun Voltage kVolts 15 Injection Voltage kVolts 1 6 NY Run Time sec 1330 PreRun Time sec 180 Injection Time sec 15 Data Delay sec 1 gt Advanced Options 10900TA Figure 5 The Setup an Instrument Protocol window 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 6 Choose Red for the Dye Color Enter 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 Dashboard Edit Library Resources x Manage Plates Assays File Name Conventions Results Group ail Analyze Instrument Protocols Dye Sets Basecalling Protocols Sizecalling Protocols QC Protocols Sequencing Analysis Protocols MicroSeqlD Protocols Fragment Analysis Protocols HID Analysis Protocols visi E 8 DB f f 4 Library Maintenance Tools Manage v Preferences Help Log Out PB Create A Edit J Duplicate elete a Setup a Size Standard
15. nalyzer 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 MSI Analysis System with the Applied Biosystems 3500 Genetic Analyzers and POP 7 polymer Some optimization may be required for individual research needs L 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 MSI Analysis System EEE 2 To set up an Instrument Protocol navigate to the Library select Instrument Protocols from the navigation pane and choose Fragment as the Filter Choose the default protocol that is the 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 8 capilllary 3500 Genetic Analyzer with a 50cm array and POP 7 polymer choose FragmentAnalysis50_POP7_1 and select Duplicate Edit the copy of this Instrument Protocol and fill in the drop down menus as appropriate for your configuration Make sure you choose Fragment as the Application Type
16. ptimal thermal cycling conditions can result in imbalanced profiles Contact Promega Technical Services at genetic promega com for assistance with other thermal cyclers 3 Materials to Be Supplied by the User e MSI Analysis System Version 1 2 Cat MD1641 e PowerPlex Matrix Standards 3100 3130 Cat 4 DG4650 MSI Analysis System E 4 Spectral Calibration Using the PowerPlex Matrix Standards 3100 3130 The PowerPlex Matrix Standards 3100 3130 Cat DG4650 contain matrix fragments labeled with four fluorescent dyes Fluorescein JOE TMR and CXR Once generated the spectral calibration file is applied during collection of MSI Analysis System data 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 TBD022 or the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide The quality of formamide is critical Use Hi Di formamide Freeze formamide in 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 avoi
17. sign sample names to wells 14 12 In the lower left portion of the screen Figure 12 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 Dashboard Edit v Library Maintenance Tools Manage Preferences Help v Log Out Plate Name ES New Plate v OpenPlate v HE Save Plate 4 Close Plate W Import iP Export 2 Find Replace View Plate Grid Report c Print v Fg EE Table View D Setup E Show In Wells v Select Wells v F Array Selection gt Column Zoomin Zoom Out Fit Define Plate Properties Assign Plate Contents a Run Instrument gt pa Preview Run tre hii Review Results View Sequencing Results View Fragment HID Results _ F Fragment DA Name MSI Experiment Barcode gt NS 4 o I Assays File Name Conventions Results Groups a Actions vY Actions v Actions EA O MI Analysis C Promega File Name C MSiResutts 9 s v 8 E 3 Link Plate for Run S oO Figure 12 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
18. sis Protocols Chemistry Matrix Standard mA Dye Set Template F Template N Arrange Dyes Dye Selection Reduced Selection Calibration Peak Order 4 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 1 j Minimum Quality Score 0 95 1 if oe Figure 2 The Create New Dye Set window 9322TA c To perform the spectral calibration with the Promega 4 dye chemistry choose Maintenance from the Menu bar then choose Spectral in the navigation pane Under the Calibration Settings choose Matrix Standard as the Chemistry Standard and P 8 p 8 1y 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 3500 Data Collection Software por Dashboard Edit Library Maintenance Tools Manage Preferences Help Log Out XK Maintenance b Print Q r Calibrate Calibration Settings Current Instrument Consumables Spatial 0 Polymer Type POP Capillary Length 50cm SC Number of Wells 96 96 FastTube 384 Chemistry Standard Matrix Standard 7 Start Run r F Performance Check Plate Posi
19. sis Range Sizing Range fui Sizing Start Size 0 Sizing Stop Size 100000 ihe roen ua Minimum Peak Height E 175 175 Common Settings Minimum Peak Half Width Peak Window Size Polynomial Degree Slope Threshold Peak Start Slope Threshold Peak End Size Calling Method Primer Peak Red v Purple 5 175 Figure 7 The Create New Sizecalling Protocol window 10 10903TA 5 To create a new Assay navigate to the Library Select Assays then select Create In the Assay window Figure 8 select the application type Fragment and the appropriate Instrument Protocol and Sizecalling Protocol Assign a descriptive assay name then select Save and Close Edit Y Dashboard View Audit History View E Signature History File Name Conventions Setup an Assay Results Group lli Anatvze ee i Instrument Protocols Dye Sets Size Standards Assay Name MSL50cm_POP7 Locked Color Application Type Protocols OROROORO Basecalling Protocols Do you wish to assign multiple instrument protocols to this assay No Yes Instrument Protocol Sizecalling Protocol GeneMapper Protocol Fo Edit Date Modified 19 May 2009 06 3 19 May 2009 06 3 19 May 2009 06 3 19 May 2009 06 3 19 May 2009 06 3 19 May 2009 06 3 19 May 2009 06 3 19 May 2009 06 3 06 Jul 2012 02 45 Figure 8
20. 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 1 Ifyou 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 e Library Maintenance Tools Manage Preferences Help v Log Out E Edit Existing Plate Common Operations yO pied we biosystems Create Create Plate New Plate from Template POP7 Polymer ABC Anode CBC Cathode 50cm 24 cap Array 192 4 ov Pip 3 3 4 2 yu i ye ee 5 4 288 2 gt 4 P sb Ls Pe 384 7i a7 f z 336 Samples Remaining 687 Days Remaining 17 Injections Remaining Instrument 3500 Instrument Laser On EP Off 48 Injections Remaining State Oven Oven Door eo mat 6 87 Days Remaining 48 Injections Remaining View Instrument Sensor Details Oven Temperature C 35 3 Detection Cell Temperature C 47 1 Pre Heat the Oven 2 Injections Performed Set Temperature to o 2 co anena lt Instrument Door Consumables Information Refresh Name Status Part Number POP 336 Samples Remaining 18 Jan 2013 06 4393708 ABC 6 87 Days Remaining 08 Jun 2013 07 4393927 CBC 6 87 Days Remaining 08 Jun 2013 07 4408256 158 Injections Remainin 10 Jun 2013
21. tion A B Dye Set Promega 4Dye v 0 Sequencing Install Standard J Allow Borrowing lt Status Ready Fragment Install Standard HID install Standard Y Capillary Run Data Q p Maintenance Wizards Capillary j j3 4 js je 7 j j jw ju j jg ju as as u jg j9 jo ja j2 j3 ja J Mns ee Planned Maintenance Run 1 Notifications Log Run 2 l Run 3 Service Log Overall n one p Passed E Failed Borrowed Not Calibrated Main Workflow Quality Value Condition Status Message j k v Intensity vs Scan Number Calibrated Data v C 0 4000 8000 12000 16000 20000 24000 28000 32000 y A Intensity vs Scan Number v Intensity vs Pixel Number E E Aee AK 0 10 20 30 40 506 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 Intensity vs Pixel Number 11093TA Figure 3 Calibration Run MSI Analysis System m E 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 soft
22. ware 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 TBD022 Capillary Run Data EEEEEA Ea Passed m Failed fa 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 Calibrated Data E 0 400 800 1200 1600 2000 2400 2800 3200 2800 2400 2000 1600 1200 200 400 Intensity vs Scan Number b 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 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 label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 5 A Sample Preparation Before preparing the samples preh
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