PowerPlex® Matrix Standards, 3100/3130, Technical Bulletin
Contents
1. Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD022 Printed in USA Page 12 4 062. 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 M atrix 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 IV B Step 3 c The peak height values must be above 750rfu and below saturation Promega Corporation 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 4 06 Page 9 Oo Promega 6 Sdect OK to close the window 7 If a capillary fails calibration it will automatically be 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 the 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 the spectral calibration should be repeated 8 When repeating the spectral calibration run samples in the same plate may be re injected a few times There will be instrument to instrument variation in the sensitivity o
3. www promega com Printed in USA Part TBD022 4 06 Page 1 oO Promega calibration should be performed on dye set Z using dye set F parameters For data collection software Version 2 0 or 3 0 the spectral calibration should be performed on dye set F Once generated this fileis applied during sample detection to calculate the spectral overlap between the four different dye colors A matrix should be generated for each individual instrument The PowerPlex Matrix Standards 3100 3130 was devdoped for use with the PowerPlex 1628 9 Y and ES Systems and can also be used with any of the GenePrint Fluorescent STR Systems A matrix should be generated for each individual instrument Protocols for operation of the ABI PRISM 3100 3100 Avent 3130 or 3130xl Genetic Analyzer should be obtained from the manufacturer Technical Manuals and additional product information are available upon request from Promega or on the Internet at www promega com Il 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 125ml N ucleaseFree Water Storage Conditions Store all components at 20 C in a non frost free freezer The fragments in the matrix standards are light sensitive and must be stored in the dark We strongly recommend that these be stored with post amplificat
4. Dye Set Z Spectral Run Module Spect36_POP4PowerPlex Spectral Parameters MtxStd Genescan_SetZPowerPlex 8 Select OK This new plate record will appear in the pending plate records table on the plate setup page of the data collection software IV E Spectral Calibration Run 1 In the pending plate records table click once on the name of the plate record you just created 2 Oncethe 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 the 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 IV 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 Sdect 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
5. PowerPlex Y System 50reactions DC6761 200 reactions DC6760 PowerPlex 1 2 System 100 reactions DC6101 PowerPlex ES System 100 reactions DC6731 400 reactions DC6730 Not for Medical Diagnostic Use Accessory Components Product Size Cat Nuclease Free Water 50ml P1193 For Laboratory Use Promega Corporation 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 1 06 Part TBD022 Page 11 STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other patents issued to the M ax Planck Gesellschaft zur F rderung der Wissenschaften e V Germany The development and use of STR loci are covered by U S Pat No 5 364 759 Australian Pat No 670231 and other pending patents assigned to Baylor College of Medicine Houston Texas Use of Promega s STR Systems requires performance of the polymerase chain reaction PCR which is the subject of European Pat Nos 201 184 and 200 362 and U S Pat Nos 4 683 195 4 965 188 and 4 683 202 owned by Hoffmann La Roche Purchase of Promega s STR Systems does not include or provide a license with respect to these patents or any other PCR related patent owned by Hoffmann La Roche or others Users of Promega s STR Systems may therefore be required to obtain a patent license depending on the country in which the systems are used For more spedfic informatio
6. 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 calibrations c Inthe Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select Spectral Calibration in the A pplication 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 d In the spectral calibration plate editor dialog box place 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 contains a sample name Select OK e Run your plate as described in the instrument user s manual f Upon completion of the run check the status of the spectral run in the Event Log window For the ABI PRISM 3100 and 3130x Genetic Analyzers we recommend that a minimum of 12 of the 16 capillaries pass calibration For the ABI PRISM 3100 Avant and 3130 Genetic Analyzers we recommend that a minimum of three of the four capillaries pass calibration If fewer than the recommended numbers of capillaries pass the spectral calibration should be repeat
7. sample or adjust the injection time or voltage based on the sensitivity of your instrument Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU are required Promega Corporation 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 4 06 Page 3 2 Run the spectral calibration procedure as described in the ABI PRISM 3100 3100 A vant 3130 or 3130xl Genetic Analyzer User s Manual with the following exceptions 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 In the Protocol Manager under Instrument Protocols select N ew Type a name for your protocol Select Spectral in the Type drop down list select F in the DyeSet drop down list and select 36 in the Array Length drop down list 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
8. Technical Bulletin PowerPlex M atrix Standards 3100 3130 INSTRUCTIONS FOR USE OF PRODUCT DG4650 IMPORTANT We recommend using this product once and then discarding it PRINTED IN USA ao MALMI Pareno AF 9TBD022 0406TBD022 PowerPlex M atrix Standards 3100 3130 All technical literature is available on the Internet at www promega com tbs 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 techserv promega com lo DESAI ON scccsccccceesssssecsscersecrecreenrtisimntiiiiniiiiimnii 1 II Product Components and Storage Conditions esses 2 III Instrument Preparation and Spectral Calibration Using D ata Collection Software Version 2 0 or Version 3 0 ABI PRISM 3100 3100 Avant 3130 and 31301 G enetic Analyzers 2 A Matrix Sample Preparati Oissar 3 B ilnsmument Preparati OMe a xtec taste lactaavactectsass artsessatatteeantsncapetisarncee dis recasticaceee 3 IV PowerPlex Spectral Calibration File Generation Using Data Collection Software Version 1 0 1 or Version 1 1 ABI PRISM 3100 and 3100 Avant G enetic AmalyZers sssccsssssssesssssssssssssssssnsesssssseeessnes 5 Ax Spectral Run MOGUL Esnia aa aAA n 5 B Spectral Parameters inriineini anana a aant ace 6 C Matix Sample Preparat Osssissnasim noname 6 Di instrument Prepar
9. a com Part TBD022 Printed in USA Page 6 4 06 Oo Promega IV B Spectral Parameters 1 To customize the spectral parameters locate the parameter file at My Computer 3100files D A ppliedBio Support Files Data Collection Support Files Calibration Data Spectral Calibration ParamFiles 2 Select MtxStd Genescan_SetF This will open the par file in Microsoft N otepad 3 Change the condition Bounds range to 4 0 12 0 4 Select File Save As to save the parameter file as anew name for example MtxStd Genescan_ SetZPowerPlex par Use this as the parameter file for all spectral calibration runs using the PowerPlex M atrix Standards 3100 3130 Note The condition number C value obtained when generating a spectral calibration will vary with the instrument A fter 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 calibrations IV C Matrix Sample Preparation There will be instrument to instrument variation in the sensitivity of detection You may need to dilute each matrix sample or adjust the injection time or voltage depending on the sensitivity or each ABI PRISM 3100 or 3100 Avant Genetic Analyzer Peak heights in the range of 1 000 4 000RFU are ideal Peak heights above 750RFU are required 1 Thaw the concentrated matrix reagents Mix the reagents by vortexing
10. at Osansa aa 7 E Spedral Cali Brati Ot RUM sdecceseSesstc cece dasa tteetdaayexrete da niaaa aaaea 8 F Spectral Calibration ReSuItS ssssssssessssessssssssssssecssssssssssesssssssessssessesssseesssesss 8 V Troubleshooting eesssssesssscsssssssssscessssseeseeesssnsmeessssnnsessssnnaeeeesseesnnies 9 VI Related Products 0 ccccsssssssssssssssssssssssssssssssssssssssssssssecssssssssssssseessssssesstssses 10 I Description Proper generation of a spectral calibration file is critical to evaluate multicolor systems with the ABI PRISM 3100 3100 Avant 3130 and 3130xI Genetic Analyzers The PowerPlex M atrix Standards 3100 3130 includes individual fragments labeled with four different fluorescent dyes Each matrix fragment is in a separate tube one tube contains a fragment labeled with fluorescein FL one tube contains a fragment labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxyfluorescein JOE one tube contains a fragment labeled with carboxy tetramethyl rhodamine TMR and one tube contains a fragment labeled with carboxy X rhodamine CXR These matrix fragments are mixed and used on the ABI PRISM 3100 3100 Avent 3130 or 3130xl Genetic Analyzer to perform a spectral calibration on a specified dye set For data collection software Versions 1 0 1 and 1 1 the spectral Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516
11. ed Promega Corporation 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 4 06 Oo Promega g When repeating the spectral calibration run samples in the same plate may be re injected a few times There will be instrument to instrument variation in the sensitivity of detection it may necessary to adjust the module injection voltage and or the injection time 5789TA Figure 1 PowerPlex M atrix Standards 3100 3130 on the ABI PRISM 3130 Genetic Analyzer using Foundation Data Collection V ersion 3 0 Figure is of the Red Yalow Blue and Green peaks in the raw data profile from each of the four capillaries in the Capillaries Viewer Promega Corporation 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 4 06 Page5 oO Promega IV PowerPlex Spectral Calibration File Generation Using D ata 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 the data collection software Versions 1 0 1 and 1 1 For instructions to gene
12. ediately on crushed ice or in an ice water bath for 3 minutes Denature the samples just prior to loading the instrument IV D Instrument Preparation These instructions apply only when using the data collection software Version 1 0 1 or 1 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 2 Open the ABI PRISM 3100 data collection software 3 Open anew plate record Name the plate and select Spectral Calibration Select 96 well as the plate type Select Finish 4 Complete the plate record spreadsheet for the wells you have loaded Enter appropriate information into the Sample N ame column 5 In the dye set column select Z from the pull down menu 6 In the spectral run module column select Spect36_POP4PowerPlex from the pull down menu This is the module that was created in Section IV A Promega Corporation 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 8 4 06 Oo Promega 7 Inthe spectral parameters column select mtxStd Genescan_SetZ PowerPlex from the pull down menu This is the file that was created in Section IV B The resulting spectral plate record should have the following settings
13. f detection it may be necessary to adjust the module injection voltage and or the injection time V Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail techserv promega com Symptoms Causes and Comments Fewer than the recommended Matrix standards are too dilute Matrix samples number of capillaries pass the that are too dilute will result in low peak spectral calibration heights If the matrix peak heights are below 1 000RFU decrease the dilution of each fragment in Step 2 of Section II A or IV C Matrix standards are 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 intense gt 6 000RFU increase the dilution of each fragment in Step 2 of Section III C or IV A and repeat the spectral calibration run Another option is to decrease the injection time so less sample is injected Promega Corporation 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 4 06 Oo Promega VI Related Products Product Size Cat PowerPlex 16 System 100 reactions DC6531 400 reactions DC6530
14. ion reagents and used separately with different pipettes tube racks etc We recommend using this product once and then discarding it II Instrument Preparation and Spectral Calibration Using D ata Collection Software Version 2 0 or Version 3 0 ABI PRISM 3100 3100 Avant 3130 and 3130xI G enetic Analyzers Materials to Be Supplied by the User Hi Di formamide A pplied Biosystems Cat 4311320 e dry heating block water bath or thermal cycler e crushed ice or an ice water bath e 3100 or 3130 capillary array 36cm Performance Optimized Polymer 4 POP 4 for the 3100 or 3130 e 10X gendiic analyzer buffer with EDTA MicroAmp optical 96 wall plate e aerosol resistant pipette tips Promega Corporation 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 2 4 06 Oo Promega ILA Matrix Sample Preparation There will be instrument to instrument variation in the sensitivity of detection Matrix sample dilutions described here may need to be optimized in individual laboratories depending on the sensitivity of each ABI PRISM 3100 3100 Avant 3130 or 3130xl Genetic Analyzer 1 Thaw the concentrated matrix reagents Mix the reagents by vortexing to 5 10 seconds before each use Do not centrifuge the matrix reagents as this may cause the DNA to be concentrated at the bottom of
15. n on obtaining a PCR icense please contact Hoffmann La Roche nthe U S effective March 29 2005 the above primary U S Pat Nos 4 683 195 4 965 188 and 4 683 202 will expire In Europe effective March 28 2006 the above primary European Pat N os 201 184 and 200 362 will expire U S Pat N os 6 238 863 and 6 767 703 have been issued to Promega Corporation for materials and methods for identifying and analyzing intermediate tandem repeat DNA markers Other patents are pending OU S Pat Nos 5 843 660 6 479 235 and 6 221 598 Australian Pat No 724531 Canadian Pat No 2 118 048 Korean Pat No 290332 Singapore Pat N o 57050 and Japanese Pat No 3602142 have been issued to Promega Corporation for multiplex amplification of STR loci Other patents are pending 2006 Promega Corporation All Rights Reserved GenePrint and PowerPlex are registered trademarks of Promega Corporation ABI PRISM and MicroAmp are registered trademarks of Applera Corporation Hi Di and POP 4 are trademarks of Applera Corporation Microsoft is a registered trademark of Microsoft Corporation 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 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
16. rate a spectral calibration using the data collection software Version 2 0 or 3 0 see Section III Materials to Be Supplied by the User Hi Di formamide A pplied Biosystems Cat 4311320 e dry heating block water bath or thermal cycler e crushed ice or an ice water bath e 3100 capillary array 36cm Performance Optimized Polymer 4 POP 4 for the 3100 instruments e 10X genetic analyzer buffer with EDTA MicroAmp optical 96 wall plate e aerosol resistant pipette tips IV A Spectral Run Module 1 Open the ABI PRISM 3100 data collection software 2 Under Tools open the module editor Select the calibration tab and then select the Spect36_POP4DefaultM odule 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 as a new name 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 will be instrument to instrument variation in the sensitivity of detection The injection times and voltage may have to be modified depending on the sensitivity of each ABI PRISM 3100 or 3100 Avant Genetic Analyzer Promega Corporation 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 promeg
17. the tube 2 Initial dilution of concentrated fragments Before mixing the dye fragments dilute an aliquot of each dye fragment 1 10 in Nuclease Free Water Vortex 5 10 seconds to mix JOE FL TMR CXR Concentrated Dye 5ul 5ul 5ul 5ul Nuclease Free Water 45ul 45ul 45ul 45ul 3 Fragment mix using 1 10 dilutions of dye fragments After the initial dilution in Step 2 mix the dye fragments as listed below Vortex each dye fragment before adding it to the HiDi formamide Component Volume Hi Di formamide 480 JOE from initial dilution 5 0ul FL from initial dilution 5 Qul TMR from initial dilution 5 0ul CXR from initial dilution 5 0ul 4 On the ABI PRISM 3100 and 3130x Genetic Analyzer 16 walls are used for matrix detection on 16 capillaries wells A1 through H2 of a 96 well tray Vortex and load 25 of the fragment mix prepared in Step 3 into each of the 16 wells On the ABI PRISM 3100 A vant or 3130 Genetic Analyzer four wells are used for matrix detection on the four capillaries wells Al through D1 in a 96 well plate Load 25ul of the fragment mix into each of the four wells 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 the samples just prior to loading the instrument II1 B Instrument Preparation 1 Prepare the matrix samples as previously described in Section III A Note You may need to dilute each matrix
18. to 5 10 seconds before each use Do not centrifuge the matrix reagents as this may cause the DNA to be concentrated at the bottom of the tube 2 Initial dilution of concentrated fragments Before mixing the dye fragments dilute an aliquot of each dye fragment 1 10 in Nuclease Free Water Vortex to mix JOE FL TMR CXR Concentrated Dye 5ul 5ul Sul Sul Nuclease Free Water 45ul 45ul 45ul 45ul Promega Corporation 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 4 06 Page 7 Oo Promega 3 Fragment mix using 1 10 dilutions of dye fragments After the initial dilution in Step 2 mix the dye fragments as listed below Vortex each dye fragment before adding it to the HiDi formamide Component Volume Hi Di formamide 480ul JOE from initial dilution 5 0ul FL from initial dilution 5 0ul TMR from initial dilution 5 0ul CXR from initial dilution 5 0ul 4 On the ABI PRISM 3100 Genetic Analyzer 16 wells are used for matrix detection on 16 capillaries wells A1 through H2 of a 96 well tray Vortex and load 25ul of the fragment mix prepared in Step 3 into each of the 16 wells On the ABI PRISM 3100 Avant Genetic Analyzer four wells are used for matrix detection on the four capillaries wells Al1through D1 in a 96 well plate 5 Denature samples at 95 C for 3 minutes then chill imm
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