Complete Protocol
Contents
1. Select 96 well as the plate type Select Finish 3 Complete the plate record spreadsheet for the wells you have loaded Enter appropriate information into the Sample Name column 4 In the Dye Set column select Z from the pull down menu 5 In the Spectral Run Module column select Spect36_POP4PowerPlex from the pull down menu This is the module that was created in Section 5 A 6 In the Spectral Parameters column select mtxStd Genescan_SetZ PowerPlex from the pull down menu This is the file that was created in section 5 B The resulting spectral plate record should have the following settings Dye Set Z Spectral Run Module Spect36_POP4PowerPlex Spectral Parameters MtxStd Genescan_SetZPowerPlex 7 Select OK This new plate record will appear in the pending plate records table on the plate setup page of the data collection software Promega Corporation gt 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD022 Printed in USA Page 16 Revised 6 13 5 E Spectral Calibration Run 1 In the pending plate records table click once on the name of the plate record you just created 2 Once the plate record is highlighted select the plate graphic that corresponds to the plate on the autosampler that contains your spectral calibration samples 3 When the plate record is linked to th2. or Reject not shown Note Refer to the 3500 Series Data Collection Software Version 1 0 HID User Manual for the criteria recommended by Applied Biosystems when accepting or rejecting a spectral calibration Capillary Run Data Eo a C E C C C E ECC ECC E EE T Passed E Failed L Borrowed Not Calibrated Capillary 1 Run 1 Quality Value 0 988753 Condition 5 685121 Status Passed Message q 0 989 5 685 Intensity vs Scan Number Calibrated Data C Beat Ra ees 0 400 800 1200 1600 2000 2400 2800 3200 o 7800 2400 2000 1600 1200 800 400 Intensity vs Scan Number b Intensity vs Pixel Number Figure 5 The Capillary Run Data display Note Some Applied Biosystems 3500 and 3500xL Genetic Analyzers show imbalance in peak heights e g peaks in the red and yellow dye channels are higher than those in the blue and green dye channels You may choose to optimize the dilution of each dye separately to obtain balanced matrix standard peaks Promega Corporation gt 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD022 Revised 6 13 Page 9 9324TA Promega 4 Instrument Preparation and Spectral Calibration Using Data Collection Software Version 2 0 or Version 3 0 ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 and 3130x Genetic Analyzers M
3. 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 Limit Scans To 2500 Sensitivity 0 4 Minimum Quality Score 0 95 Figure 3 Create New Dye Set Promega Corporation Toll Free in USA 800 356 9526 2800 Woods Printed in USA Revised 6 13 Before Scan 5000 Hollow Road Phone 608 274 4330 Fax 608 277 2516 Madison WI 53711 5399 USA WWW promega com Part TBD022 Page 7 9322TA Promega 3 B Instrument Preparation continued c To perform the spectral calibration with the Promega 4 dye chemistry go to the Maintenance tab select Spectral and under the Calibration Run tab choose the appropriate fields Choose Matrix Standard from the Chemistry Standard pull down menu and the new Promega 4 dye set created in Step 2b from the Dye Set pull down menu Figure 4 d Select Start Run E 3500 Data Collection Software oes Dashboard Edit Y Library Maintenance Tools Manage Preferences Help Log Out BE Maintenance SS ee Pen ere Te peseearcn wew SoechaliCalbatonRenct ie Pi port St ral Calibration F alts j ignature view Spectral Calibrati eport g Print Qe Calibrate Calibration Settings Current Instrument Consumables Spatial oO Polymer Type POP4 Capillary Length 36cm p gt Spectr Number of Wells 96 96 FastTube
4. 384 Chemistry Standard Matrix Standard gt start Run FA Performance Check Jf Plate Position A OB Dr Set Promega 4Dye hd 0 Sequencing Install Standard W Allow Borrowing Status Ready Fragment Install Standard HID Install Standard Capillary Run Data a Maintenance Wizards i 2 Bo ju 5 ie jis jo 20 21 23 P Planned Maintenance ae ewa Notifications Log Service Log Schedule 3 i 5 T o Passed E Failed E Borrowed Wot Calibrated Main Workflow Quality Value Condition Status Message Ai i Intensity vs Scan Number a n E oo Raw Data ey SS Se A 0 4000 8000 12000 16000 20000 24000 28000 32000 Tao 50000 Y S A d E lt 7 ae 4 40000 1 J SS 30000 AWN 4 z gt 20000 10000 7 p Intensity vs Scan Number gt Intensity vs Pixel Number Figure 4 Calibration Run Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD022 Printed in USA Page 8 Revised 6 13 Promega 3 If fewer than the recommended number of capillaries pass the spectral calibration run will be repeated automatically up to three times Upon completion of the spectral calibration check the quality of the spectral in the Capillary Run Data display Figure 5 and choose either Accept
5. bottle of polymer fresh buffer and water and a capillary array with fewer than 100 injections All capillaries failed spectral Monitoring fragment migration in the calibration Capillaries Viewer during the spectral calibration run can provide information that might be useful for troubleshooting purposes Reinject the spectral calibration plate and monitor the Capillaries Viewer during the run Note any unusual peak formations or extremely high or low peak heights Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD022 Revised 6 13 Page 19 Promega 7 Related Products Product Size Cat PowerPlex 16 HS System 100 reactions DC2101 400 reactions DC2100 PowerPlex 16 System 100 reactions DC6531 400 reactions DC6530 PowerPlex CS7 System 100 reactions DC6613 PowerPlex S5 System 100 reactions DC6951 400 reactions DC6950 Not for Medical Diagnostic Use Accessory Components Product Size Cat Nuclease Free Water 50ml P1193 For Laboratory Use 2006 2009 2010 2013 Promega Corporation All Rights Reserved GenePrint and PowerPlex are registered trademarks of Promega Corporation ABI PRISM Applied Biosystems and MicroAmp are registered trademarks of Applied Biosystems Hi Di is a trademarks of Applera Corporation POP 4 is a registered trademark of Life Technologi
6. 274 4330 Fax 608 277 2516 www promega com Part TBD022 Printed in USA Page 6 Revised 6 13 9247TA Promega 2 To perform a spectral calibration with the Promega 4 dye chemistry a new dye set should be created If a new dye set already has been created proceed to Section 3 B Step 2 c a To create this new dye set navigate to the Library highlight Dye Sets and select Create b The Create a New Dye Set tab will appear Figure 3 Name the Dye Set select Matrix Standard for the Chemistry and select F Template for the Dye Set Template Select Save i Dashboard Edit Library Resources e View Audit History P Library Maintenance Tools Manage Y Preferences Help Log Out View E Signature History K Manage Plates Assays File Name Conventions Results Group hd Search ae ili Analyze Instrument Protocols Size Standards Basecalling Protocols Sizecalling Protocols fc Protocols Sequencing Analysis Protocols MicroSeqID Protocols Fragment Analysis Protocols HID Analysis Protocols Chemistry Standard Calibration Date Setup a Dye Set Dye Set Name Promega 4Dye Chemistry Matrix Standard v Dye Set Template F Template g lt g Arrange Dyes Dye Selection Reduced Selection Calibration Peak Order 4 w Parameters Capillary Array Serial Number Is Signed Locked
7. in individual laboratories depending on the sensitivity of each ABI PRISM 3100 or 3100 Avant or Applied Biosystems 3130 or 3130x Genetic Analyzer The optimal dilution may differ for each dye fragment You also may need to adjust injection time or voltage in Section 4 B to achieve a passing spectral calibration Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU and below the saturation point of the instrument are required Promega Corporation gt 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD022 Printed in USA Page 14 Revised 6 13 The same plate of matrix standards can be re injected up to four times To re inject the same matrix standards plate add an injection by selecting Plate Manager then Edit Select Edit again in the top left corner of the window then select Add single run 1 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 2 Initial dilution of concentrated fragments Before combining the matrix standards dilute the individual matrix standards 1 10 in Nuclease Free Water Vortex for 5 10 seconds to mix FL JOE TMR CXR Concentrated Dye 5ul 5ul Byul 5ul Nuclease Free Water 45ul 45 ul A5ul 45ul 3 Fragment
8. mix using 1 10 dilutions of matrix standards After the initial dilution in Step 2 combine the 1 10 dilutions as directed below Vortex for 5 10 seconds to mix Component Volume Hi Di formamide 480ul FL from initial dilution 5 0ul JOE from initial dilution 5 0ul TMR from initial dilution 5 0ul CXR from initial dilution 5 0ul Note Differences in instrument sensitivity may result in peak imbalance or reduced peak height You may need to prepare a new plate and adjust the dilution of individual matrix dyes Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU and below the saturation point of the instrument are required Use the worksheet below to record the dilutions used with your capillary electrophoresis instrumentation Component Volume Hi Di formamide FL from initial dilution JOE from initial dilution TMR from initial dilution CXR from initial dilution 4 On the ABI PRISM 3100 Genetic Analyzer 16 wells are used for spectral calibration on 16 capillaries wells A1 through H2 of a 96 well plate Load 25ul of the fragment mix prepared in Step 3 into each of the 16 wells Briefly centrifuge the plate to remove bubbles Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD022 Revised 6 13 Page 15 Promega 5 C Matrix Sample Preparation contin
9. w m gt gt Create Create Plate Edit Existing View New Plate from Template Plate Run Results Quick Start Run Quick View e Gauges POP4 Polymer ABC Anode CBC Cathode 36cm 24 cap Array 192 5 Bi a 3 al i s 5 64 6 CO T AAA a T AEE 1 i at ei a w ii a ki ao s ia I A r 6 I gt f 6 A 9 i gt 38 a a S I4 Ca 0 j 4384 0 47 0 7 on A 160 eo eo 48 Samples Remaining 7 Days Remaining 7 Days Remaining 66 Injections Performed 6 Injections Remaining 46 Injections Remaining 46 Injections Remaining Pre Heat the Oven Instrument 3500 Instrument State Idle View Instrument Sensor Details Laser On Oven On Set Temperature to Oven Temperature C 60 0 Detection Cell Temperature C 49 96 60 eal C Start Pre Heat EP Off OvenDoor Close Instrument Door Close Consumables Information Comani me O O porone opone tne poe O Polymer POP4 48 Samples Remaining 5 06 Jan 2011 06 1006009 4393715 Anode Buffer ABC 7 Days Remaining 0 18 Mar 2011 07 1006014 4393927 Cathode Buffer CBC 7 Days Remaining 0 23 Mar 2011 07 1007021 4403256 Capillary Array 36cm 24 cap J4 Injections Remaining 51 03 Feb 2011 02 08 4404687 Serial M309K0803 Maintenance Notifications 7 Rane pe pe pO o p O O Figure 2 The Dashboard Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608
10. 22 Printed in USA Page 2 Revised 6 13 These matrix fragments are mixed and used on the ABI PRISM 3100 or 3100 Avant or Applied Biosystems 3130 3130xl 3500 or 3500xL Genetic Analyzer to perform a spectral calibration for a specified dye set For Data Collection Software versions 1 0 1 and 1 1 the spectral calibration should be performed on dye set Z using dye set F parameters For Data Collection Software version 2 0 and 3 0 the spectral calibration should be performed on dye set F The instructions for use with the Applied Biosystems 3500 and 3500xL Genetic Analyzers were generated with 3500 Data Collection Software v1 0 HID on an Applied Biosystems 3500xL Genetic Analyzer A spectral calibration file is generated for a specific set of dyes array type 4 8 16 or 24 capillaries and array length 36cm Once generated this file is applied during sample detection to calculate the spectral overlap between the four different dye colors and separate the raw fluorescent signals into individual dye signals The PowerPlex Matrix Standards 3100 3130 was developed for use with the PowerPlex 16 16 HS 55 and CS7 Systems and the PowerPlex 16 and ES Monoplex Systems and can be used with any of the GenePrint Fluorescent STR Systems A matrix should be generated for each individual instrument Protocols to operate the fluorescence detection instruments should be obtained from the manufacturer Technical Manuals and additional produc
11. 3130xl 3500 and 3500xL Genetic Analyzers The PowerPlex Matrix Standards 3100 3130 includes individual fragments labeled with four different fluorescent dyes Figure 1 Each matrix fragment is provided in a separate tube one tube contains a fragment labeled with fluorescein FL one tube contains a fragment labeled with 6 carboxy 47 5 dichloro 2 7 dimethoxyfluorescein JOE one tube contains a fragment labeled with carboxy tetramethylrhodamine TMR and one tube contains a fragment labeled with carboxy X rhodamine CXR oCapibary Date Mon Aug 14 155822 CDT 2008 ADI es Dyo Sai F x Lass Acie Calbration for Crys Sat F Meis used 10r Copdiery 1 4 Won Anas 11 18 55 22 COT 2008 Corti BAH A Vax o sans23 Ugi of Calkeations tor Dye Set F por Aug 11 155822 COT 2006 gt e Hi EIE lt Sytem Saus Bun a0 20000 1206_ HOT stabus hoe chengedto Compisiad 5 ice a State B Sorvics Corson EB ortir D v32 DA o l6am 5 Figure 1 PowerPlex Matrix Standards 3100 3130 on the Applied Biosystems 3130 Genetic Analyzer using Data Collection Software version 3 0 This figure shows the CXR red TMR yellow JOE green and FL blue peaks in the spectral viewer from one of the four capillaries Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD0
12. TECHNICAL BULLETIN PowerPlex Matrix Standards 3100 3130 Instructions for use of Product DG4650 Note The PowerPlex Matrix Standards 3100 3130 can be used to perform spectral calibration on the Applied Biosystems 3500 and 3500xL Genetic Analyzers e Pro Revised 6 13 TBD022 PowerPlex Matrix Standards 3100 3130 All technical literature is available on the Internet at www promega com protocols Please visit the web site to verify that you are using the most current version of this Technical Bulletin Please contact Promega Technical Services if you have questions on use of this system E mail genetic promega com Ti TS CB ENON ete tetas soe ccs cc astaneence E esses ares tears serene teens 2 2 Product Components and Storage Conditions ssssssesssesessseesssseerseee 3 3 Instrument Preparation and Spectral Calibration Using the Applied Biosystems 3500 and 3500xL Genetic Analyzers Fame b Grote 104 od ed oF o 1410 Wren pea mene no eevee re Pr er re arse mere 4 D Jiomament i ren basta 0 4 renee tere erm Tere crere rarer A AR 6 4 Instrument Preparation and Spectral Calibration Using Data Collection Software Version 2 0 or Version 3 0 ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 and 3130x1 Genetic Analyzers 0 0 0 ccccesessesseseeesseseeseceeneesesteneeeneens 10 A Matrix Sample PreparatiON neh cect roseacescse sence cts ccoeieeg aes sea
13. aterials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath e centrifuge compatible with 96 well plates e 3100 or 3130 capillary array 36cm e performance optimized polymer 4 POP 4 polymer for the 3100 or 3130 e 10X genetic analyzer buffer with EDTA e MicroAmp optical 96 well plate e aerosol resistant pipette tips Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze formamide in D 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 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 ABI PRISM 3100 or 3100 Avant or Applied Biosystems 3130 or 3130x Genetic Analyzer The optimal dilution may differ for each dye fragment You also may
14. contains a sample name Select OK f Run your plate as described in the instrument user s manual g Upon completion of the run check the status of the spectral run in the Event Log window For the ABI PRISM 3100 and Applied Biosystems 3130xl Genetic Analyzers we recommend that a minimum of 12 of 16 capillaries pass calibration For the ABI PRISM 3100 Avant and Applied Biosystems 3130 Genetic Analyzers we recommend that a minimum of three of four capillaries pass calibration If fewer than the recommended numbers of capillaries pass repeat the injection see Section 4 B Step 2 If the spectral calibration still does not pass repeat the spectral calibration 5 PowerPlex Spectral Calibration File Generation Using Data Collection Software Version 1 0 1 or Version 1 1 ABI PRISM 3100 and 3100 Avant Genetic Analyzers Note To create a successful spectral calibration two file modifications must be made the first involves the run module and the second involves the parameter file These instructions only apply to Data Collection Software versions 1 0 1 and 1 1 For instructions to generate a spectral calibration using Data Collection Software version 2 0 or 3 0 see Section 4 Materials to Be Supplied by the User e Hi Di formamide Applied Biosystems Cat 4311320 e 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath e centrifuge compatible with 96 well plates e 3100 capilla
15. e for example Spect36_POP4PowerPlex Use this as the initial run module for all spectral calibration runs using the PowerPlex Matrix Standards 3100 3130 Note There may be instrument to instrument variation in the sensitivity of detection You may have to modify the injection time or voltage depending on the sensitivity of each instrument 5 B Spectral Parameters 1 To customize the spectral parameters locate the parameter file at My Computer 3100files D AppliedBio Support Files Data Collection Support Files Calibration Data Spectral Calibration ParamFiles 2 Select MtxStd Genescan_SetF This will open the par file in Microsoft Notepad 3 Change the condition bounds range to 4 0 12 0 4 Select File Save As to save the parameter file with a new name for example MtxStd Genescan_ SetZPowerPlex par Use this as the parameter file for all spectral calibration runs using the PowerPlex Matrix Standards 3100 3130 Note The condition number C value obtained when generating a spectral calibration will vary with the instrument After obtaining a spectral calibration that performs acceptably the condition bounds range in the previous step may be narrowed to more critically evaluate C values for subsequent spectral calibrations 5 C Matrix Sample Preparation There may be instrument to instrument variation in the sensitivity of detection The dilutions described here may need to be optimized
16. e Array Length drop down list e Select the spectral module you created in the previous step in the Run Module drop down list Finally select Edit Parameters Change the lower condition bound to 4 0 and change the upper condition bound to 12 0 Select OK in the Edit Parameters window and select OK in the Protocol Manager Note The condition number C value obtained when generating a spectral calibration will vary with the instrument After obtaining a spectral calibration that performs acceptably the condition bounds range in the previous step may be narrowed to more critically evaluate C values for subsequent spectral calibrations d In the New Plate Dialog screen create a new plate record as described in the instrument user s manual In the dialog box that appears select Spectral Calibration in the Application drop down list and select 96 well as the plate type Add entries in the owner and operator windows name the plate and select OK Promega Corporation gt 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD022 Printed in USA Page 12 Revised 6 13 e In the Spectral Calibration Plate Editor Dialog box record sample names in the appropriate cells In the Instrument Protocol column select the protocol you created in Step 2 b Ensure that this information is present for each row that
17. e assigned the spectral data from the nearest passing capillary to the left To achieve an effective spectral calibration file we recommend that a minimum of 12 out of 16 capillaries pass calibration for the ABI PRISM 3100 Genetic Analyzer For the ABI PRISM 3100 Avant Genetic Analyzer we recommend that a minimum of three of the four capillaries pass calibration If fewer than the recommended number of capillaries pass repeat the injection To re inject the same matrix standards plate add an injection by selecting Plate Manager then Edit Select Edit again in the top left corner of the window then select Add single run The same plate of matrix standards can be re injected up to four times If the spectral calibration still does not pass repeat the spectral calibration Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD022 Revised 6 13 Page 17 Promega 6 Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com 6 A Applied Biosystems 3500 and 3500xL Genetic Analyzers Symptoms Causes and Comments Fewer than the recommended Matrix standards were too dilute Matrix number of capillaries passed the samples that are too dilute wi
18. e plate the plate graphic will change from yellow to green the plate record moves from the pending plate records table to the linked plate records table and the Run Instrument button becomes enabled 4 Select the Run Instrument button on the toolbar to start the spectral calibration run 5 F Spectral Calibration Results 1 After the spectral calibration run is completed the Spectral Calibration Result window opens indicating which capillaries passed calibration A indicates the capillary passed An X indicates the capillary failed 2 Select OK 3 Review the spectral calibration data In the Tools menu of the data collection software select Display Spectral Calibration 4 Select the Dye Set button select Dye Set Z from the pull down menu and select OK 5 The Matrices for Dye Set Z window displays the spectral calibration data for each capillary Use the scroll bar to review the data for each of the 16 capillaries For the PowerPlex Matrix Standards 3100 3130 the following conditions must be met for the capillary to pass calibration a The Q value must be greater than 0 95 b The condition number or C value must fall within the range previously determined in Section 5 B Step 3 c The peak height values must be above 750RFU and below the saturation point of the instrument 6 Select OK to close the window 7 Ifa capillary fails calibration it will automatically b
19. educed peak heights You the spectral calibration may need to adjust injection time or voltage to achieve a passing spectral calibration The same plate of matrix standards can be re injected up to four times Alternatively you may need to prepare a new plate and adjust the dilution of individual matrix dyes Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU and below the saturation point of the instrument are required Matrix standards were too dilute Matrix samples that are too dilute will result in low peak heights If matrix peak heights are below 1 000RFU decrease the dilution of each fragment in Step 2 of Section 4 A or 5 C Matrix standards were too concentrated Matrix sample peak heights that are too high may result in bleedthrough or oversubtraction in other dye colors which may result in spectral calibration failure If matrix sample peak heights are too high gt 6 000RFU increase the dilution of each fragment in Step 2 of Section 4 A or 5 C and repeat the spectral calibration run Another option is to decrease the injection time so that less sample is injected Incomplete denaturation can cause extra peaks in one or all color channels Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to loading the capillary Do not cool samples in a thermal cycler or 20 C freezer For best spectral calibration results use a fresh
20. er container with 1X running buffer e MicroAmp optical 96 well plate and septa e Hi Di formamide Applied Biosystems Cat 4311320 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 avoid 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 For additional information on performing spectral calibration refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 3 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 The optimal dilution may differ for each dye 1 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 2 Initial d
21. es Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products Promega Corporation gt 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD022 Printed in USA Page 20 Revised 6 13
22. ilution of concentrated fragments Before combining the matrix standards dilute the individual matrix standards 1 10 in Nuclease Free Water as described below Vortex for 5 10 seconds to mix FL JOE TMR CXR Matrix Standard 5ul 5ul Byul 5ul Nuclease Free Water 45ul 45 ul 45ul 45ul Promega Corporation gt 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD022 Printed in USA Page 4 Revised 6 13 3 Fragment mix using 1 10 dilutions of matrix standards After the initial dilution in Step 2 combine the 1 10 dilution of each matrix standard as directed below Vortex for 5 10 seconds to mix Component Volume Hi Di formamide 668ul FL from initial dilution 8 0ul JOE from initial dilution 8 0ul TMR from initial dilution 8 0ul CXR from initial dilution 8 0ul Note Differences in instrument sensitivity may result in peak imbalance or reduced peak height You may need to prepare a new plate and adjust the dilution of individual matrix dyes Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU and below the saturation point of the instrument are required Use the worksheet below to record the dilutions used with your capillary electrophoresis instrumentation Component Volume Hi Di formamide FL from initial dilution JOE from initial dilution TMR from initial dilution CXR from initial dilution 4 O
23. ll result in low spectral calibration peak heights Decrease the dilution of each fragment in Step 2 of Section 3 A Matrix standards were too concentrated Matrix samples that are too concentrated may result in bleedthrough or oversubtraction in other dye colors which may result in spectral calibration failure Increase the dilution of each fragment in Step 2 of Section 3 A Samples were not denatured completely Incomplete denaturation can cause extra peaks in one of all color channels Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to loading the capillary Do not cool samples in a thermal cycler or 20 C freezer All capillaries failed spectral Monitoring fragment migration in the Capillaries calibration Viewer during the spectral calibration run can provide information that might be useful for troubleshooting purposes Note any unusual peak formations or extremely high or low peak heights Promega Corporation gt 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD022 Printed in USA Page 18 Revised 6 13 6 B ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 and 3130x Genetic Analyzers Symptoms Causes and Comments Fewer than the recommended Differences in instrument sensitivity may result number of capillaries passed in peak imbalance or r
24. n the Applied Biosystems 3500xL Genetic Analyzer only wells A1 to H3 of the 96 well plate are used for spectral calibration Load 25y11 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 only wells A1 to H1 of the 96 well plate are used for spectral calibration Load 25pl 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 6 Place the plate in the 3500 series 96 well standard plate base and cover with the plate retainer Place the plate assembly in Position A on the autosampler with the labels facing you Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD022 Revised 6 13 Page 5 Promega 3 B Instrument Preparation 1 Set the oven temperature to 60 C then select Start Pre Heat at least 30 minutes prior to the first injection to preheat the oven Figure 2 De i i H Library Maintenance Tools Manage Preferences Help Log Out Common Operations
25. need to adjust injection time or voltage in Section 4 B to achieve a passing spectral calibration Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU and below the saturation point of the instrument are required The same plate of matrix standards can be re injected up to four times To re inject the same matrix standards plate add an injection by selecting Plate Manager then Edit Select Edit again in the top left corner of the window then select Add single run 1 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 Promega Corporation gt 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD022 Printed in USA Page 10 Revised 6 13 2 Initial dilution of concentrated fragments Before combining the matrix standards dilute the individual matrix standards 1 10 in Nuclease Free Water Vortex for 5 10 seconds to mix FL JOE TMR CXR Concentrated Dye Byul 5ul 5ul Byul Nuclease Free Water 45ul 45ul 45ul 45ul 3 Fragment mix using 1 10 dilutions of matrix standards After the initial dilution in Step 2 combine the 1 10 dilutions as directed below Vortex for 5 10 seconds to mix Component Volume Hi Di formamide A480ul FL from i
26. nitial dilution 5 0ul JOE from initial dilution 5 0ul TMR from initial dilution 5 0ul CXR from initial dilution 5 0ul Note Differences in instrument sensitivity may result in peak imbalance or reduced peak height You may need to prepare a new plate and adjust the dilution of individual matrix dyes Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU and below the saturation point of the instrument are required Use the worksheet below to record the dilutions used with your capillary electrophoresis instrumentation Component Volume Hi Di formamide FL from initial dilution JOE from initial dilution TMR from initial dilution CXR from initial dilution 4 On the ABI PRISM 3100 and Applied Biosystems 3130x Genetic Analyzers 16 wells are used for spectral calibration on 16 capillaries wells A1 through H2 of a 96 well plate Load 25ul of the fragment mix prepared in Step 3 into each of the 16 wells Briefly centrifuge the plate to remove bubbles On the ABI PRISM 3100 Avant and Applied Biosystems 3130 Genetic Analyzers four wells are used for matrix detection on the four capillaries wells A1 through D1 of a 96 well plate Load 25ul of fragment mix in each of the four wells 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 instrumen
27. nneece ss ettceseametecde 10 De ASTRO CRG IRE Pat atl Ol eorias E E 12 5 PowerPlex Spectral Calibration File Generation Using Data Collection Software Version 1 0 1 or Version 1 1 ABI PRISM 3100 and 3100 Avant Genetic Analyzers ce 13 R E E E 10 EA A E E E A E TS 14 Mies n E a o e E A E E E E E E 14 C Matrix Sample Preparation sssssssssessssssssessssressssresssssrresssrressstreessnreensssrressserees 15 D JS PUIG I TEP ata OW sereias r venation ce 16 E Spectral Calibration RUN cence ceec ete acescapenccesosconc tee coedeaseacoececetacdeartoreeecaeaceyecacteey 17 Pear e EE Noy e2 ufo e bcs eee eer ee ene E E ean ne ere ree ee 17 Bs TROUBLES hooting essenin iere aieia iaon e Eiir eeni 18 A Applied Biosystems 3500 and 3500xL Genetic Analyzers s es 18 B ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130 and 3130x Genetic ANaly ZELS sssossisrsesistirsijarino inisesin 19 7 Related Products ssessessssesessssssssssrressssseessssrressssrreesssreesssrrresssrreensssreesss 20 Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD022 Revised 6 13 Page 1 Promega 1 Description Proper generation of a spectral calibration file is critical to evaluate multicolor systems with the ABI PRISM 3100 and 3100 Avant and Applied Biosystems 3130
28. ry array 36cm e performance optimized polymer 4 POP 4 polymer for the 3100 instruments e 10X genetic analyzer buffer with EDTA e MicroAmp optical 96 well plate e aerosol resistant pipette tips 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 avoid 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 Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD022 Revised 6 13 Page 13 Promega 5 A Spectral Run Module 1 Open the ABI PRISM 3100 Data Collection Software 2 In the Tools menu open the Module Editor Select the Calibration tab then select the Spect36_POP4DefaultModule module 3 Confirm or change the settings for the following Inj kV 3 Inj Secs 5 Data Delay Time 100 Run Time 800 seconds 4 Select Save As and save the module with a new nam
29. t Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD022 Revised 6 13 Page 11 Promega 4 B Instrument Preparation 1 Prepare the matrix samples as previously described in Section 4 A Note Differences in instrument sensitivity may result in peak imbalance or reduced peak height You may need to adjust injection time or voltage to achieve a passing spectral calibration Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU and below the saturation point of the instrument are required 2 Perform the spectral calibration as described in the ABI PRISM 3100 or 3100 Avant or Applied Biosystems 3130 or 3130x Genetic Analyzer user s manuals with the following modifications a In the Module Manager select New Select Spectral in the Type drop down list and select Spect36_POP4 in the Template drop down list Confirm or change the following settings Inj kV 3 Inj Secs 5 Data Delay Time 100 Run Time 800 seconds b Select Save As and save the module with a new name c In the Protocol Manager under Instrument Protocols select New Type a name for your protocol Make the following selections in the Protocol Editor e Spectral in the Type drop down list e F in the DyeSet drop down list e 36 in th
30. t information are available at www promega com 2 Product Components and Storage Conditions Product Cat PowerPlex Matrix Standards 3100 3130 DG4650 Not for Medical Diagnostic Use Includes 25ul Fluorescein Matrix 25ul JOE Matrix 25ul TMR Matrix 25ul CXR Matrix e 1 25ml Nuclease Free Water Storage Conditions Store all components at 30 to 10 C in a nonfrost free freezer Do not store reagents in the freezer door where the temperature can fluctuate The fragments in the matrix standards are light sensitive and must be stored in the dark We strongly recommend that the matrix standards be stored with post amplification reagents and used separately with different pipettes tube racks etc Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD022 Revised 6 13 Page 3 3 Instrument Preparation and Spectral Calibration Using the Applied Biosystems 3500 and 3500xL Genetic Analyzers Materials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath e centrifuge compatible with 96 well plates e aerosol resistant pipette tips 3500 3500xL capillary array 36cm e performance optimized polymer 4 POP 4 polymer in a pouch for the 3500 or 3500xL e anode buffer container with 1X running buffer e cathode buff
31. ued On the ABI PRISM 3100 Avant Genetic Analyzer four wells are used for spectral calibration on four capillaries wells A1 through D1 of a 96 well plate Load 25 ul of the fragment mix in each of the four wells Briefly centrifuge the plate to remove bubbles 5 Denature samples at 95 C for 3 minutes then chill immediately on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument 5 D Instrument Preparation These instructions apply only when using the Data Collection Software version 1 0 1 or 1 1 Refer to the instrument user s manual for instructions on cleaning the pump blocks installing the capillary array performing a spectral calibration and adding polymer to the reserve syringe Differences in instrument sensitivity may result in peak imbalance or reduced peak height The dilutions described here may need to be optimized in individual laboratories depending on the sensitivity of each ABI PRISM 3100 or 3100 Avant Genetic Analyzer The optimal dilution may differ for each dye fragment You also may need to adjust injection time or voltage in Section 5 C to achieve a passing spectral calibration Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU and below the saturation point of the instrument are required 1 Open the ABI PRISM 3100 Data Collection Software 2 Open a new plate record Name the plate and select Spectral Calibration
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