Complete Protocol
Contents
1. 608 274 4330 Fax 608 277 2516 5 www promega com TMD046 Revised 10 15 Promega 4 B Instrument Preparation We have found that the use of fresh polymer and a new capillary array results in an optimal spectral calibration Representative data are shown in Figure 3 ile View Service Tools Wizards License 6 Dye Help gt Boh amp 4 amp Bie R OP AR gt Bat t BGA Instruments GA Instruments gt ga3130 gt DELLHPPGBY 18D gt Spectral Viewer Results Group at Database Manager 3 823130 1 0 _ Plate Manager 08 T Protocol Manager j Module Manager 0 6 Run History 0 4 CET Viewer ElEventLog 0 2 a Instrument Protocol 0 0 Hd Spatial Calbration Vie 50 100 150 200 250 Ba capllary Viewer Intensity vs Pixel Number E Cap Array Viewer E Spectral Calibration V 1600 fp Reextracton 1200 CJ DELLHPPGBY 180 Einstrument Status 800 SEPT Chart eget Che 400 IE JEvent Log Bd Spatial Run Schedules 0 Run Scheduler EMEI 400 hpPiate view 0 1000 2000 3000 4000 5000 BBRun View Sameer es Intensity vs Scan Number aap Capikaries Vemar Capillary Data Wed Jun 25 09 34 04 COT 2014 BB Cap Array Viewer y ete AD1 Ah Manual Control service Log Dye Set v Active Calibration for Dye Set 36 Matrix used for Capillary 1 i Condition 6 710472 Q Value 0 99625 List of Calibrations for Dye Set 36 Figure 3 Representative data for the PowerPlex 6C Matrix Standard on the Applied Biosystems 3130 Genetic2. EEA Minimum Quality Score 0 95 Notes Oy NS Changed after scan to 800 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD046 Revised 10 15 www promega com Promega 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 and choose either Accept or Reject Note Refer to the 3500 Series Data Collection Software User Manual for the criteria recommended when accepting or rejecting a spectral calibration Instrument Preparation and Spectral Calibration Using the Applied Biosystems 3130 and 3130xl Genetic Analyzers with Data Collection Software Version 4 0 with the DC v4 6 Dye Module v1 License Materials to Be Supplied by the User centrifuge compatible with 96 well plates aerosol resistant pipette tips 3130 3130xl capillary array 36cm POP 4 polymer for the 3130 or 3130xl 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa Hi Di formamide Applied Biosystems Cat 4311320 For additional information on performing spectral calibration refer to the Applied Biosystems 3130 3130xl Genetic Analyzer User Guide 4 A Matrix Sample Preparation 1 At the first use thaw the 6C Matrix Mix
3. for the Dye Set Template Under Parameters change the After Scan number to 800 from the default number of 500 Select Save c To perform the spectral calibration go to the Maintenance tab select Spectral and under the Calibration Run tab choose the appropriate fields Choose Matrix Standard from the Chemistry Standard drop down menu and the new Promega 6 color dye set i e Promega J6 created in Step 2b from the Dye Set drop down menu d Select Start Run Dashboard Edit v Library Resources SA Create 4 Edit Fj Duplicate fj Delete Import p Export poy E Signature View Audit History View E Signature History ARB Fie biosystems f Edit Dye Set Promega J6 Manage Setup a Dye Set Plates Assays File Name Conventions Results Group Dye Set Name Promega_J6 lilis Analyze Chemistry Matrix Standard Instrument Protocols Dye Set Template J6 Template Dye Sets Size Standards naii Dye Selection See Reduced Selection Sizecalling Protocots Calibration Peak Order 6 4 3 a QC Protocols Sequencing Analysis Protocols ann The parameters will be used for instruments configured with 36cm capillary array and polymer POP4 MicroSeqID Protocols R ae ne Matrix Condition Number Upper Limit 8 0 Fragment Analysis Protocols j i Locate Start Point After Scan 800 Before Scan 5000 HID Analysis Protocols eae A A Limit Scans To 2750 si Sensitivity 0 4 V WEGE
4. Analyzer with Data Collection Software Version 4 0 with the DC v4 6 Dye Module v1 License using POP 4 polymer 1 Set the oven temperature to 60 C and preheat the oven for at least 15 minutes prior to the first injection 2 To perform a spectral calibration for Promega 6 color PowerPlex Systems a new Run Module and Protocol should be created If a new Run Module and Protocol were created previously proceed to Step 2 d a Inthe Run Module Editor select New Select SPECTRAL in the Type drop down list and select Spect36_POP4 in the Template drop down list Change the Data Delay Time to 400 and the Run Time to 800 Change the Injection Time to 6 seconds See Figure 4 6 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD046 Revised 10 15 www promega com Promega Run Module Editor en CaS Run Module Description Name 1 2kV6sec_400delay800run Type SPECTRAL Template Spect36_POP4 Description Run Module Settings Name Value Range 60_ 18 65 Deg C Poly_Fill_Vol 4840 4840 38000 steps Current_Stability 50 0 2000 uAmps PreRun_Voltage 15 0 0 15KVolts Pre_Run_Time 180 1 1000 sec Injection_Voltage 1 2 1 15 KVolts Injection_Time 6 1 600sec Voltage_Number_Of_Steps 40 1 100 nk Voltage_Step_Interval 15 1 60 sec Data_Delay_Time 400 1 3600 sec Run_Voltage 15 0 0 15 KVolts R
5. Instrument Proparal oTi epsiscen coves os enesuses sseaeica news seeeuaesdeveessvscdaneiosesesencabavtorserseuiummacensdensieabsereiecssens 6 De OUD FCS OMI Sass avaceenbates secsanstavces S ET A S E 8 Biis SPCCUT Al CAIN ALCON OR E E E E E A E E EA 8 5 B Spectral Calibration on the Applied Biosystems 3130 and 3130xl Genetic Analyzers ce00e 9 Ge Summary of Chant Seer aAa E NNa aa 10 1 Description Proper generation of a spectral calibration file is critical to evaluate multicolor systems with the Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers The PowerPlex 6C Matrix Standard consists of DNA fragments labeled with six different fluorescent dyes FL 6C JOE 6C TMR 6C CXR 6C TOM 6C and WEN in one tube The spectral calibration is performed using the J6 dye set Once generated the spectral calibration file is applied during sample detection to calculate the spectral overlap and separate the raw fluorescent signals into individual color signals The PowerPlex 6C Matrix Standard was developed for use with the Promega 6 color PowerPlex Systems and is compatible with the Applied Biosystems 3500 and 3500xL Genetic Analyzers as well as Applied Biosystems 3130 and 3130x Genetic Analyzers with Data Collection Software Version 4 0 with the DC v4 6 Dye Module v1 License Life Technologies A protocol to operate the Applied Biosystems 3130 3130x 3500 or 3500xL Genetic Analyzer should be obtained fro
6. Peaks in multiple dye channels were detected prior to the orange peak Increase the data delay time to exclude those peaks and re inject the matrix standard The data delay time specified for the Applied Biosystems 3130 or 3130x Genetic Analyzer was too long and the orange peak was not captured during data collection Decrease the data delay time and re inject the matrix standard Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 9 www promega com TMD046 Revised 10 15 Promega 6 Summary of Changes The following changes were made to the 10 15 revision of this document 1 Expired legal disclaimers were removed 2 The WEN dye name was changed to WEN from WEN 6C Patent Pending ITMR 6C CXR 6C TOM 6C and WEN dyes are proprietary 2015 Promega Corporation All Rights Reserved PowerPlex is a registered trademark of Promega Corporation Applied Biosystems and MicroAmp are registered trademarks of Applied Biosystems Hi Di is a trademark of Applera Corporation POP 4 is a registered trademark of Life Technologies 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
7. access the Promega online catalog for the most up to date information on Promega products 10 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD046 Revised 10 15 www promega com
8. and Matrix Dilution Buffer completely After the first use store the reagents at 2 10 C Vortex the 6C Matrix Mix for 10 15 seconds prior to use Add 10ul of the 6C Matrix Mix to one tube of Matrix Dilution Buffer Vortex for 10 15 seconds Note the date of dilution on the tube The diluted 6C Matrix Mix can be stored for up to 1 week at 2 10 C Add 10ul of the diluted 6C Matrix Mix prepared in Step 2 to 500ul of Hi Di formamide Vortex for 10 15 seconds For the Applied Biosystems 3130x Genetic Analyzer 16 wells are used for spectral calibration on 16 capillaries wells A1 through H2 of a 96 well plate Add 15ul of the 6C Matrix Mix with formamide prepared in Step 3 to each of the 16 wells After placing the septa on the plate briefly centrifuge the plate to remove bubbles Do not heat denature For the Applied Biosystems 3130 Genetic Analyzers four wells are used for spectral calibration on four capillaries wells A1 through D1 in a 96 well plate Add 15ul of the 6C Matrix Mix with formamide prepared in Step 3 to each of the four wells After placing the septa on the plate briefly centrifuge the plate to remove bubbles Do not heat denature Place the plate in the 3130 series 96 well standard plate base and cover with the plate retainer Do not start the spectral calibration run until the oven is preheated to 60 C Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
9. 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 7 www promega com TMD046 Revised 10 15 Promega 4 B Instrument Preparation continued 6 Run your plate as described in the instrument user s manual 7 Upon completion of the run check the status of the spectral calibration in the Event Log window For the Applied Biosystems 3130x Genetic Analyzers we recommend that a minimum of 12 of the 16 capillaries pass calibration For the 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 spectral calibration 5 Troubleshooting 5 A Spectral Calibration This section provides general information about spectral calibration For information specific to the Applied Biosystems 3130 and 3130x Genetic Analyzers see Section 5 B 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 Symptoms Causes and Comments Fewer than the recommended number of Poor quality formamide was used The quality of formamide is capillaries passed the spectral calibration critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that comp
10. TECHNICAL MANUAL PowerPlex 6C Matrix Standard Instructions for Use of Product DG4900 All technical literature is available at www promega com protocols Visit the web site to verify that you are using the most current version of this Technical Manual E mail Promega Technical Services if you have questions on use of this system genetic promega com t GSCI OM s5ecsaugeedecesacosannus cess sewenncoteccecessage lt eagueasasteseseisestoscsececeateeoucsseastusnuseressspueusassesmecuedtectsevatexeess 1 2 Product Components and Storage Conditions ccsccssccssscsscessccsccesccesccsscesccesccssccescenscsccescessceesceees 2 3 Instrument Preparation and Spectral Calibration Using the Applied Biosystems 3500 and 3500xL Genetic AnalyZers ccssccsscosscosscssccsscesscesccesccscesccesccsceescees 2 3 A Matrix Sample Preparation oasasecesiacsnaeaueaeunecnedacacasacadasesacaacassvceuvaeane ees ens aeuseduderasarudcuayaeneasacaeneaeses 2 2B ATSIPUMENE Preparat ioli sscscscespersaasasecdescinnetcncunvenseisnenssesiursudeusdeesdcucsatsdacuesesanaausecovesdauadeareenddansiees 3 4 Instrument Preparation and Spectral Calibration Using the Applied Biosystems 3130 and 3130x Genetic Analyzers with Data Collection Software Version 4 0 with the DC v4 6 Dye Module v1 License 5 AA Matrix Sample Preparation sxcsaseiereressiceciesviewieasncashisasiatieaseteseds sccas sac andseaan ease meareaaaseddiviereiasinteaass 5 AB
11. Version 4 0 with the DC v4 6 Dye Module v1 License For general trouble shooting information about spectral calibration see Section 5 A S toms Causes and Comments Fewer than the recommended number of Peak heights for the matrix standard were too low Matrix peak capillaries passed the spectral calibration heights must be a minimum of 750RFU to pass spectral calibration Increase the injection time Peak heights for the matrix standard were too high gt 5 000RFU Decrease the injection time Check the Event Log in the Instrument Status screen for a reason for the failure e g bad dye order or too many spectral calibration candidates Check the raw data view of the failed capillaries in the Spectral Viewer failed capillaries are shaded tan in the plate diagram Look for signs of low signal high signal or baseline noise before the matrix peaks Adjust the run conditions as described below and re inject the matrix standard If the cause for failure is unclear after viewing the Event Log and Spectral Viewer monitoring fragment migration in the Capillaries Viewer during spectral calibration run can provide information that will be useful for troubleshooting purposes Re inject the matrix standard and monitor the Capillaries Viewer during the run Note any unusual peak formations or extremely low or high peak heights Based on the information obtained while watching the Capillaries Viewer you may need to adjust the run conditions
12. ed Capillary 8 Run1 Quality Value 0 997186 Condition 5 492412 Status Passed Message q 0 997 c 5 492 v Intensity vs Scan Number Calibrated Data Ee ES A 0 400 800 1200 1600 2000 2400 2800 3200 Intensity vs Scan Number v Intensity vs Pixel Number Be Bol Be 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 Intensity vs Pixel Number Figure 1 Representative data for the PowerPlex 6C Matrix Standard on the Applied Biosystems 3500xL Genetic Analyzer using POP 4 polymer and Data Collection Software Version 2 0 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 3 www promega com TMD046 Revised 10 15 Promega 3 B Instrument Preparation continued 1 Set the oven temperature to 60 C and then select the Start Pre Heat icon at least 30 minutes prior to the first injection to preheat the oven 2 To perform a spectral calibration for the Promega 6 color PowerPlex Systems a new dye set should be created If a new dye set was created previously proceed to Step 2c To create the new dye set navigate to the Library highlight Dye Sets and select Create b The Create a New Dye Set window will appear Figure 2 Name the Dye Set i e Promega J6 select Matrix Standard for the Chemistry and select J6 Template
13. ete with DNA during injection which results in lower peak heights and reduced sensitivity Matrix standard was too dilute Matrix standard that is too dilute will result in low spectral calibration peak heights which may result in spectral calibration failure Increase the volume of diluted 6C Matrix Mix added to the Hi Di formamide during sample preparation Matrix standard was too concentrated Matrix standard that is too concentrated may result in spectral calibration failure due to saturated peaks bleedthrough or oversubtraction in other dye colors Decrease the volume of diluted 6C Matrix Mix added to the Hi Di formamide during matrix sample preparation Reboot the CE instrument and the instrument s computer Repeat the spectral calibration Ensure that the oven is preheated to 60 C prior to spectral calibration All capillaries failed spectral calibration For best spectral calibration results use fresh polymer fresh buffer and water and a capillary array with fewer than 100 injections 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD046 Revised 10 15 www promega com Promega 5 B Spectral Calibration on the Applied Biosystems 3130 and 3130xl Genetic Analyzers The following information is specific to spectral calibration on the Applied Biosystems 3130 and 3130x Genetic Analyzers with Data Collection Software
14. ibration Using the Applied Biosystems 3500 and 3500xL Genetic Analyzers Materials to Be Supplied by the User centrifuge compatible with 96 well plates aerosol resistant pipette tips 3500 3500xL capillary array 36cm POP 4 polymer for the 3500 or 3500xL anode buffer container with 1X buffer cathode buffer container with 1X buffer MicroAmp optical 96 well plate and septa Hi Di formamide Applied Biosystems Cat 4311320 For additional information on performing spectral calibration refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 3 A Matrix Sample Preparation 1 At the first use thaw the 6C Matrix Mix and Matrix Dilution Buffer completely After the first use store the reagents at 2 10 C 2 Vortex the 6C Matrix Mix for 10 15 seconds prior to use Add 10ul of the 6C Matrix Mix to one tube of Matrix Dilution Buffer Vortex for 10 15 seconds Label the tube with the date of dilution The diluted 6C Matrix Mix can be stored for up to 1 week at 2 10 C 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TMD046 Revised 10 15 www promega com Promega 3 Add 10ul of the diluted 6C Matrix Mix prepared in Step 2 to 500ul of Hi Di formamide Vortex for 10 15 seconds 4 For the Applied Biosystems 3500xL Genetic Analyzer wells Al through H3 of the 96 well plate are
15. m the manufacturer A spectral calibration must be generated for each individual instrument A new matrix should be run after major maintenance on the system such as changing the laser calibrating or replacing the CCD camera or changing the polymer type or capillary array We also recommend that you generate a new matrix after the instrument is moved to a new location In some instances a software upgrade may necessitate generation of a new matrix Individual labs should determine the frequency of matrix generation Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 www promega com TMD046 Revised 10 15 1 2 Product Components and Storage Conditions PRODUCT SIZE CAT PowerPlex 6C Matrix Standard 5 preps DG4900 Not For Medical Diagnostic Use Includes e 150ul 6C Matrix Mix e 5 x 200ul Matrix Dilution Buffer Storage Conditions Upon receipt store all components at 30 C to 10 C in a nonfrost free freezer After the first use store the PowerPlex 6C Matrix Standard components at 2 10 C We strongly recommend that the PowerPlex 6C Matrix Standard be stored with post amplification reagents The PowerPlex 6C Matrix Standard is light sensitive dilute the 6C Matrix Mix in the Matrix Dilution Buffer in the provided amber tube Store the diluted 6C Matrix Mix at 2 10 C for up to 1 week 3 Instrument Preparation and Spectral Cal
16. un_Time 800 300 14000 sec Ok Cancel Figure 4 Run Module Editor settings b Name the Run Module e g Promega J6 and select OK c Inthe Protocol Manager under Instrument Protocols select New Type a name for your protocol e g Promega J6 d Make the following selections in the Protocol Editor Spectral in the Type drop down list J6 in the DyeSet drop down list POP4 for the polymer 36 in the Array Length drop down list Matrix Standard for the chemistry 2 Select the spectral module you created in the previous step in the Run Module drop down list Select Edit Parameters and change the Minimum Quality Score Q value to 0 95 2 Select OK in the Edit Parameters window and select OK in the Protocol Editor 3 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 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 4 Inthe spectral calibration plate editor dialog box place sample names in the appropriate cells 5 Inthe Instrument Protocol column select the protocol you created in Step 2 Ensure that this information is present for each sample Select OK Promega Corporation 2800 Woods Hollow Road Madison WI 53711
17. used for spectral calibration Add 15ul of the 6C Matrix Mix with formamide prepared in Step 3 to each of the 24 wells After placing the septa on the plate briefly centrifuge the plate to remove bubbles Do not heat denature For the Applied Biosystems 3500 Genetic Analyzer wells A1 through H1 of the 96 well plate are used for spectral calibration Add 15ul of the 6C Matrix Mix with formamide prepared in Step 3 to each of the eight wells After placing the septa on the plate briefly centrifuge the plate to remove bubbles Do not heat denature 5 Place the plate in the 3500 series 96 well standard plate base and cover with the plate retainer Do not start the spectral calibration run until the oven is preheated to 60 C 3 B Instrument Preparation We have found that the use of fresh polymer and a new capillary array results in an optimal spectral calibration Representative data are shown in Figure 1 i Export Spectral Calibration Results j E gt View Spectral Calibration Report Qs Print Calibration Run Calibration Information Dye Set Chemistry Standard Calibration Date zi Capillary Array Serial Number Is Signed Matrix Standard 17 Nov 2014 12 39 02 PM M314G1415 No Matrix Standard 17 Nov 2014 12 07 25 PM M314G1415 No Capillary Run Data 2 a 2 2 ja Capillary Runi Run 2 Run 3 Overall 10 fir j2 13 ja js fjs j je jo j feii Passed B Failed Borrowed Not Calibrat
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