Complete Protocol
Contents
1. spikes Minor voltage visible in one or all of the color changes or urea crystals passing by the laser can channels cause spikes or unexpected peaks Spikes sometimes appear in one color but often are easily identified by their presence in more than one color Reinject the samples to confirm 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 TBD018 Printed in USA Page 12 Revised 8 09 5 Troubleshooting continued Symptoms Causes and Comments Poor quality matrix extra peaks CE related artifacts contaminants visible in one or all of the color Contaminants in the water used with the channels continued ABI PRISM 310 Genetic Analyzer and for diluting the 10X genetic analyzer buffer can generate peaks in the blue and green dye colors Use autoclaved water to clean the pump block and to prepare sample dilutions Change vials and wash the buffer reservoir Poor quality matrix elevated baseline Matrix used was generated on another and or inverted peaks in analyzed instrument A matrix must be generated for samples see Figure 2 each instrument Wrong dye used Generate the matrix using the same dyes as those contained in the samples Oversubtraction of signal because signal is saturated When generating a matrix avoid choosing samples with peak heights that are higher than th2. 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 TBD018 Revised 8 09 Page 9 4 D Sample Preparation and Loading J Combine 1 5pl of each matrix sample with 1 5p of Blue Dextran Loading Solution Denature each sample for 2 minutes at 95 C and immediately chill on crushed ice for 3 minutes Note Instrument detection limits vary therefore the amount of product mixed with loading cocktail may need to be increased or decreased After the 15 to 20 minute prerun pause the instrument by clicking on the Pause button When the prerun is paused the water will continue to circulate to keep the gel warm during the sample loading Use a 30cc syringe filled with buffer to flush the urea from the well area Load 1 5p1 of each denatured sample into the respective wells Place the lid on the upper buffer chamber and close the instrument door 4 E Gel Electrophoresis and Detection 1 4 After loading click on Cancel to stop the prerun Make sure that the run time is set at 3 hours then click on Run to begin electrophoresis Monitor the electrophoresis by observing the gel image and status windows Allow electrophoresis to proceed for 3 hours The largest fragment will have migrated past the laser Track and extract the gel lanes 4 F Matrix Generation for the ABI PRISM 3
3. 2 Assemble the glass plates by placing 0 2mm side gel spacers between the front and rear glass plates Hold the plates together using binder clamps 4 clamps on each side Place the assembly horizontally on a test tube rack or similar support Prepare a 5 Long Ranger acrylamide gel total of 50ml by combining the ingredients listed in Table 2 Stir the solution until the urea has dissolved Table 2 Preparation of a 5 Long Ranger Polyacrylamide Gel 8 9 Component 5 Gel Final Concentration urea 18g 6M deionized water 26ml 10X TBE 5ml 1X 50 Long Ranger gel solution 5ml 5 total volume 50ml Note Long Ranger Singel Packs can be used Caution Acrylamide Long Ranger gel solution is a neurotoxin and suspected carcinogen avoid inhalation and contact with skin Read the warning label and take the necessary precautions when handling this substance Always wear double gloves and safety glasses when working with acrylamide solutions Filter the acrylamide solution through a 0 2 micron filter e g Nalgene tissue culture filter and degas for an additional 5 minutes Add 35pl of TEMED and 2501 of fresh 10 ammonium persulfate to the 50ml of acrylamide solution and mix gently Using a disposable 30cc syringe pour the gel by starting at the well end of the plates and carefully injecting the acrylamide between the horizontal glass plates Allow the solution to fill the top width of the plates While maint
4. Promega 5 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 Unable to generate a matrix Poor capillary electrophoresis CE injection due to faint or no peaks Reinject the sample Check the syringe for leakage Check the laser power Poor quality formamide used Use only high quality formamide when running samples on the ABI PRISM 310 Genetic Analyzer The conductivity of the deionized formamide should be less than 1004S cm Samples degraded due to improper storage Store matrices in the dark at 20 C Peak heights too low Peak heights should be 1 000 4 000RFU for the ABI PRISM 310 Genetic Analyzer and 800 2 000RFU for the ABI PRISM 377 DNA Sequencer To increase peak heights increase the injection time or loading volume Samples not denatured Heat denature the samples and immediately chill on crushed ice before loading the gel or capillary Unable to generate a matrix Insufficient number of peaks Choose an area miscellaneous that includes a minimum of 5 peaks for matrix standardization With newer versions of GeneScan Analysis Software an acceptable matrix can be created with fewer than 5 peaks However for optimal results use as many peaks as possible Poor quality matrix extra peaks CE related artifacts
5. 20 C Multiple freeze thaw cycles or long term storage at 4 C can cause a breakdown of the formamide Formamide with a conductivity gt 100yS cm can contain ions that compete with DNA during injection This results in lower peak heights and reduced sensitivity A longer injection time might not increase the signal Caution Formamide is an irritant and teratogen avoid inhalation and contact with skin Read the warning label and take the necessary precautions when handling this substance Always wear double gloves and safety glasses when working with formamide Instrument Preparation 1 Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 2 Open the ABI PRISM 310 data collection software 3 Prepare a GeneScan sample sheet as described in the ABI PRISM 310 Genetic Analyzer User s Manual Enter the appropriate sample information in the sample info column Create a new GeneScan injection list Select the appropriate sample sheet by using the pull down menu 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 TBD018 Revised 8 09 Page 3 Oo Promega 3 A Instrument Preparation continued 4 Select the GS STR POP4 1ml A Module using the
6. Medical Diagnostic Use Accessory Components Product Size Cat Internal Lane Standard 600 150p1 DG1071 Fluorescent Ladder CXR 60 400 Bases 65pl DG6221 Gold ST R 10X Buffer 1 2ml DM2411 Mineral Oil 12m DY1151 Nuclease Free Water 50ml P1193 For Laboratory Use Sample Preparation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Plexor HY System 800 reactions DC1000 200 reactions DC1001 For Laboratory Use Not for Medical Diagnostic Use GenePrint Plexor and PowerPlex are registered trademarks of Promega Corporation DNA IQ is a trademark of Promega Corporation ABI PRISM GeneMapper and GeneScan are registered trademarks of Applera Corporation Hi Di and POP 4 are trademarks of Applera Corporation Liqui Nox is a registered trademark of Alconox Inc Long Ranger and Long Ranger Singel are registered trademarks of Lonza BioProducts Macintosh is a registered trademark of Apple Computer Inc Nalgene is a registered trademark of Nalge Nunc International Windows NT is a registered trademark of Microsoft Corporation 2004 2008 2009 Promega Corporation All Rights Reserved 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
7. OOM tor 2008 BE tor 2008 A Figure 2 Elevated baseline A sample was run on an ABI PRISM 310 Genetic Analyzer and analyzed using GeneScan Analysis Software The resulting electropherogram shows an elevated baseline below 270 bases An elevated baseline can be the result of using a matrix from another instrument using a matrix made on the same instrument before service or using a matrix made with different dyes 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 TBDO18 Printed in USA Page 14 Revised 8 09 m GeneScan Project 2 14 00 Display 4 y FENG o o e200 ao o00 poo goo Soo goon eoo god sooo peoo son peoo 400 200 Oise E 208 J0E matrix ig mix 1 15 a q LAJI TBM 206 J0 matrix Ig mix 1 15 dil SS ee ee ee es __ DE 20v JOE matrix lg mix 1 15 dil THI 208 J0 matrix ig mix 17150 k Y lt a A A ae E S A pane ee 2884TA03_0A Figure 3 Inverted baseline The four matrix samples from the PowerPlex Matrix Standards 310 377 were run on an ABI PRISM 310 Genetic Analyzer A matrix was made using the GeneScan Analysis Software but no start at point was entered for the matrix samples The resulting matrix was applied
8. 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 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD018 Printed in USA Page 16 Revised 8 09
9. copy of the matrix file should be stored in the ABI folder located in the system folder Anew matrix can be applied to previously run samples by highlighting the sample in the GeneScan project Under sample select install new matrix highlight the new matrix and click on open The new matrix will be applied to the sample file and the samples can be analyzed using the new matrix The quality of the matrix can be verified Apply the new matrix file to the samples used to generate the matrix Analyze the matrix samples using all 4 dye colors The matrix samples should have peaks of 800 2 000RFU in the dye colors listed in Step 3 As you evaluate each sample the baselines for the other three dye colors should be relatively flat Reuse of Glass Plates Separate the glass plates and discard the gel Clean the glass plates with hot water and a detergent such as 1 Liqui Nox detergent Rinse extremely well with deionized water and allow the plates to air dry Do not scrape the plates with abrasive materials during this process Gel extrusion gel expands into the comb during a run can occur due to a build up of residue If this occurs soak the plates in 2N HCl for 15 minutes then rinse thoroughly 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 TBD018 Revised 8 09 Page 11 Oo
10. select install new matrix highlight the new matrix and click on open The new matrix will be applied to the sample file and the samples can be analyzed using the new matrix The quality of a matrix can be verified Apply the new matrix file to the samples used to generate the matrix Analyze the matrix samples using all four dye colors The matrix samples should have peaks between 1 000 4 000RFU in the dye colors listed in Step 3 The baselines for the other three dye colors should be relatively flat A small amount of bleedthrough may be seen with the TMR yellow into the CXR red channel 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 TBD018 Page 6 Printed in USA Revised 8 09 Oo Promega 4 A Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer Materials to Be Supplied by the User Solution compositions are provided in Section 6 e dry heating block water bath or thermal cycler e crushed ice Long Ranger gel solution Lonza Cat 50611 or Long Ranger Singel pack for ABI sequencers 377 36cm Lonza Cat 50691 e 10 ammonium persulfate Cat V3131 e TEMED e Urea Cat V3171 TBE 10X buffer e Nalgene tissue culture filter 0 2 micron e aerosol resistant pipette tips e gel loading pipette tips e 36cm front and rear glass p
11. to the JOE Matrix sample file and analysis was done using all four colors The result shows inverted peaks in the blue yellow and red channels 6 Composition of Buffers and Solutions 10 ammonium persulfate Add 0 05g of ammonium persulfate to 500p1 of deionized water Use 250pl of 10 ammonium persulfate for each 50ml of acrylamide gel solution deionized formamide Use ultra pure grade formamide Applied Biosystems Hi Di Formamide Cat 431132 Use a conductivity meter to check the conductivity The deionized formamide must have a conductivity lt 100pS cm Aliquots of deionized formamide can be made and frozen Avoid multiple freeze thaw cycles Blue Dextran Loading Solution 88 25 formamide 15mg ml blue dextran 41mM EDTA pH 8 0 TBE 10X buffer 107 8g Tris base 744g EDTA Na EDTA 2H 0 55 0g boric acid Dissolve the Tris base and EDTA in 800ml deionized water Slowly add the boric acid and monitor the pH until the desired pH of 8 3 is obtained Bring the volume to 1 liter with deionized water 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 Revised 8 09 Part TBD018 Page 15 Oo Promega 7 Related Products Product Size Cat PowerPlex 1 2 System 100 reactions DC6101 GenePrint Fluorescent STR Systems 100 reactions DC5171 Not for
12. 77 DNA Sequencer I 2 Open the GeneScan project Review the raw data from the individual matrix samples Highlight the sample file name then go under the sample menu and select raw data Move the cursor beyond the primer peak so the crosshair is on a flat portion of the baseline Record the X value number shown at the bottom of the window Matrix generation requires a minimum of 5 peaks Select an area that includes 5 peaks in each color See Figure 1 With newer versions of GeneScan Analysis Software an acceptable matrix can be generated with fewer than 5 peaks However for optimal results use as many peaks as possible 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 TBD018 Page 10 Printed in USA Revised 8 09 Oo Promega 4 G Under the File Menu select New then click the matrix icon The points field should have the default value of 100 000 Click on the dye color for each matrix and indicate the sample file that corresponds to that dye Enter the recorded X value from Step 2 in the start at field Dye Color Corresponding Matrix Blue Fluorescein Matrix Green JOE Matrix A Yellow TMR Matrix Red CXR Matrix Click OK and the matrix file will be generated Save the matrix file in the Matrix Standards Folder located in the GeneScan folder A
13. Matrix Iml Blue Dextran Loading Solution Storage Conditions Store all components at 20 C The DNA 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 away from pre amplification materials and used separately with different pipettes tube racks etc Additional product information and ordering information for accessory components and related products is available upon request from Promega or at www promega com 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 TBD018 Printed in USA Page 2 Revised 8 09 oO Promega 3 A Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User Solution compositions are provided in Section 6 e dry heating block water bath or thermal cycler e crushed ice e 310 capillaries 47cm x 50pm e Performance Optimized Polymer 4 POP 4 e sample tubes and septa e aerosol resistant pipette tips e 10X genetic analyzer buffer e deionized formamide with conductivity lt 100uS cm Hi Di Formamide Applied Biosystems Cat 4311320 Note The quality of the formamide is critical Use deionized formamide with a conductivity lt 100pS cm Aliquots of formamide can be made and frozen at
14. Technical Bulletin PowerPlex Matrix Standards 310 377 INSTRUCTIONS FOR USE OF PRODUCT DG3640 PRINTED IN USA Revised 8 09 Part TBD018 PowerPlex Matrix Standards 310 377 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 1 D sripti nen iinn rnaen 1 2 Product Components and Storage Conditions cssssssseeeeessssseeeee 2 3 Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer wd A Instrument Preparation vied B Sample Preparations sisvveccsssisscsssssnsssansssassebsesacsvvessasetessobnssonssessesuieseastendbsessssiiv ene 4 C Capillary Electrophoresis and Detection sssssssssessssssssesesesesesssesesssseeseeeseees 4 D Matrix Generation for the ABI PRISM 310 Genetic Analyze 5 4 Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer scccccseeeeees ad A Polyacrylamide Gel Preparation ail B Instrument Preparation sinse na E A ANN 9 E Pernt T A S 9 D Sample Preparation and Loading 7 E Gel Electrophoresis and Detection sssssssssssssssssssssssssssssssssesssessesssees 10 F Matrix Generation for the ABI PRISM 377 DNA Sequencer sssssssee 10 G R
15. aining a constant flow of solution gently tap the glass plates to assist the movement of solution to the bottom of the plates Insert a 36 well sharkstooth comb or 34 well squaretooth comb between the glass plates Sharkstooth combs with 64 or 96 wells can also be used Secure the comb with 3 evenly spaced clamps Keep the remaining acrylamide solution as a polymerization control 10 Allow polymerization to proceed for gt 2 hours Check the polymerization control to be sure that polymerization has occurred Note The gel can be stored overnight if a paper towel saturated with deionized water and plastic wrap are placed around the top and bottom to prevent the gel from drying out crystallization of the urea will destroy the gel 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 TBD018 Page 8 Printed in USA Revised 8 09 4 B Instrument Preparation 4 C 1 2 5 Open the ABI PRISM 377 data collection software Prepare a sample sheet as described in the GeneScan Analysis Software User s Manual Enter the appropriate sample information in the sample info column Create a new GeneScan run and use the following settings Plate Check Module Plate Check A PreRun Module PR GS 36A 2400 Run Module GS 36A 2400 Collect Time 3 hours Well to Read Distance 36cm Select the appropri
16. ate sample sheet and comb selection by using the pull down menus Select none for the gel matrix file Gel Prerun i Remove the clamps from the polymerized acrylamide gel If necessary clean any excess acrylamide from the glass plates with paper towels saturated with deionized water Shave any excess polyacrylamide away from the comb and remove the comb If using a sharkstooth comb carefully insert the sharkstooth comb teeth into the gel approximately 1 2mm Position the gel glass plate unit in the 377 cassette Secure the cassette in the instrument and perform a plate check as recommended in the ABI PRISM 377 DNA Sequencer User s Manual If the horizontal line graph is not flat remove the cassette clean the plate surface and repeat plate check Add TBE 1X buffer to the top and bottom buffer chambers of the instrument Using a 30cc syringe filled with buffer remove any air bubbles and unpolymerized acrylamide from the well area of the gel and place the lid on the upper buffer chamber Using a syringe with a bent 19 gauge needle remove the air bubbles from the bottom of the gel Attach the heating plate connect the water tubing attach all electrodes close the instrument door and click on the PreRun button Allow the gel to prerun for 15 20 minutes or until the gel temperature is at least 40 C Open the Status Window to monitor the temperature of the gel Prepare the matrix samples during the gel prerun
17. e recommended RFU values as this can result in a matrix that causes inverted peaks or elevated baseline An improvement in analyzed samples can be seen by diluting the matrix samples in water before preparation for use in this protocol prior to Section 3 B Step 1 or Section 4 D Step 1 Matrix baseline has inverted peaks Incorrect or no start at value entered See Figure 3 Start at value should have a flat baseline Wrong colors assigned to the dyes Confirm the dye and color selection Fluorescein Blue JOE A Green TMR Yellow CXR Red 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 TBD018 Revised 8 09 Page 13 Oo Promega 5 Troubleshooting continued Symptoms Causes and Comments Previously generated matrix no longer Changes to or aging of instrument components performs optimally The sensitivity of the instrument can change if the instrument has been moved or recently serviced replacement or realignment of the laser CCD camera power supply or mirrors The sensitivity also can change over time due to aging of the instrument These changes can result in poor matrix performance Generate a new matrix GeneScan Project 12 28 99 Display 1 Elevated baseline
18. eset Glass Platesne n n Aan 11 Ds LPOUbLESHO ORIG sssi irsinin 12 6 Composition of Buffers and Solutions eecsssseesessssseecsssneeesssnee 15 Ts Related Productssccsssassiscccccccsssvssscocessscveccccossstvsaiiaasnnntveassennseeevecstasnsveeevvssseeeeeecd 16 1 Description Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA Sequencer To prepare a matrix four standards are run under the same capillary electrophoresis CE or gel conditions that are used for samples and allelic ladders The PowerPlex Matrix Standards 310 377 consist of DNA fragments labeled with four different fluorescent dyes one tube contains DNA fragments labeled with fluorescein one tube contains DNA fragments labeled with carboxy tetramethylrhodamine TMR two tubes contain DNA fragments labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxyfluorescein JOE and one tube contains DNA fragments labeled with carboxy X rhodamine CXR 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 TBD018 Revised 8 09 Page 1 Oo Promega 1 Description continued Use the Fluorescein Matrix JOE Matrix TMR Matrix and CXR Matrix for the blue green yellow and red standards respectively The PowerPlex Matrix Standards 310 377 includes bot
19. h JOE A and JOE B labeled fragments but this protocol uses only JOE A The PowerPlex Matrix Standards 310 377 is used with the PowerPlex 1 2 System or any of the GenePrint Fluorescent STR Systems fluorescein labeled A matrix should be generated for each individual instrument and Promega chemistry Protocols for operation of the fluorescence detecting instrumentation should be obtained from the manufacturer For information on Promega s other fluorescent STR systems refer to the GenePrint Fluorescent STR Systems Technical Manual TMD006 PowerPlex 1 1 System Technical Manual TMD008 PowerPlex 1 2 System Technical Manual TMD009 PowerPlex 2 1 System Technical Manual TMD011 PowerPlex 16 System Technical Manual TMD012 PowerPlex ES System Technical Manual TMD017 and PowerPlex Y System Technical Manual TMDO18 These Technical Manuals and additional product information are available upon request from Promega or at www promega com For the PowerPlex 16 HS PowerPlex 16 PowerPlex S5 Y and ES Systems the PowerPlex Matrix Standards 310 Cat DG4640 is required for matrix standardization for the ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA Sequencer 2 Product Components and Storage Conditions Product Cat PowerPlex Matrix Standards 310 377 DG3640 Not for Medical Diagnostic Use Includes 50pl Fluorescein Matrix 50pl JOE Matrix A 50pl JOE Matrix B 50pl TMR Matrix 50pl CXR
20. lates 36cm gel spacers 0 2mm thick e 36 well sharkstooth comb or 34 well squaretooth comb 0 2mm thick e clamps e g large office binder clamps e Liqui Nox or other detergent Polyacrylamide Gel Preparation Hazardous reagents are used in the preparation and use of gels for the ABI PRISM 377 DNA Sequencer The reagents and their hazards are listed in Table 1 Table 1 Hazardous Reagents Reagents for ABI PRISM 377 DNA Sequencer Hazard acrylamide suspected carcinogen Long Ranger gel solution toxic ammonium persulfate oxidizer corrosive formamide irritant teratogen contained in the Blue Dextran Loading Solution TEMED corrosive flammable urea irritant The following protocol is for preparation of a 36cm denaturing polyacrylamide gel for use with the ABI PRISM 377 DNA Sequencer Low fluorescence glass plates are recommended and can be obtained from the instrument manufacturer 1 Thoroughly clean the glass plates with hot water and a 1 Liqui Nox solution or another dilute laboratory detergent solution Rinse extremely well using deionized water Allow the glass plates to air dry in a dust free environment 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 TBD018 Revised 8 09 Page 7 Oo Promega 4 A Polyacrylamide Gel Preparation continued
21. les Highlight the Sample File name then go to the sample menu and select raw data Move the cursor beyond the primer peak so the crosshair is on a flat portion of the baseline Record the X value number shown at the bottom of the window Select an area that includes at least 5 peaks matrix generation requires a minimum of 5 peaks With newer versions of GeneScan Analysis Software an acceptable matrix can be generated with fewer than 5 peaks However for optimal results use as many peaks as possible See Figure 1 o TMR matrix Ig mix 1 15 dil ziz Xp 500 1000 1500 2o00 2500 2000 soo sooo asoo sooo soo sooo esoo 7o00 7s00 L gy Start At 2882TA03_0A EEEk n TZ Figure 1 TMR Matrix raw data The TMR Matrix standard was run on an ABI PRISM 310 Genetic Analyzer GeneScan Analysis Software was used to view the raw data option under sample The cursor was placed on the baseline and the start at value of 3529 was determined by using the readout in the lower left hand corner of the window 3 Under the File Menu select New then click the Matrix icon The points field should have the default value of 100 000 Click on the dye color for each matrix and indicate the sample file that corresponds to that dye Enter the X value recorded from Step 2 in the start at field Dye Color Correspo
22. nding Matrix Blue Fluorescein Matrix Green JOE Matrix A Yellow TMR Matrix Red CXR Matrix 4 Select OK and the matrix file will be generated 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 gt www promega com Printed in USA Part TBD018 Revised 8 09 Page 5 Oo Promega 3 D Matrix Generation for the ABI PRISM 310 Genetic Analyzer continued 5 Save the matrix file in the Matrix Standards Folder located in the GeneScan folder Be sure to use a meaningful name that indicates with which STR system the matrix file will be used For the Macintosh version of the software a copy of the matrix file is automatically saved in the GS Matrix folder For the Windows NT version of the software store a copy of the matrix file in the matrix folder at C appliedbio shared analysis sizecaller matrix Note To generate a matrix in GeneMapper ID open the GeneMapper Manager and click on the Matrices tab Click on the New button Select the appropriate sample files to be used for each dye color and enter the X value in the start at field as described in Steps 2 and 3 Click on the Create button then on the OK button to generate and save the matrix Anew matrix can be applied to previously run samples by highlighting the sample in the GeneScan project Under the sample menu
23. pull down menu Change the run time to 30 minutes and keep the settings for the remaining parameters as shown below Inj Secs 5 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time minutes 30 Note The injection time may need to be optimized for individual instruments 5 Select none for the matrix file 3 B Sample Preparation 1 For each matrix sample combine 21 of the matrix standard with 25p1 deionized formamide or water 2 Denature each sample for 3 minutes at 95 C and immediately chill on crushed ice for 3 minutes 3 Assemble the tubes in the appropriate autosampler tray 48 tube or 96 tube 4 Place the autosampler tray in the instrument and close the instrument doors 3 C Capillary Electrophoresis and Detection 1 After loading the sample tray and closing the doors click on the Run button to start the capillary electrophoresis system 2 Monitor the electrophoresis by observing the raw data and status windows 3 Each sample will take approximately 40 minutes for syringe pumping sample injection and sample electrophoresis 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 TBDO18 Printed in USA Page 4 Revised 8 09 3 D Matrix Generation for the ABI PRISM 310 Genetic Analyzer 1 Open the GeneScan project 2 Review the raw data from the individual matrix samp
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