PowerPlex(R) Matrix Standards, 310 Technical Bulletin, TBD021
Contents
1. Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD021 Printed in USA Page 10 Revised 8 10 The following protocol is for preparing 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 glass plates with hot water and a 1 Liqui Nox solution or another dilute laboratory detergent solution Rinse extremely well using deionized water Allow glass plates to air dry in a dust free environment 2 Assemble glass plates by placing 0 2mm side gel spacers between the front and rear glass plates Hold plates together using binder clamps four clamps on each side Place the assembly horizontally on a test tube rack or similar support 3 Prepare a 5 Long Ranger acrylamide gel total of 50ml by combining the reagents listed in Table 2 Stir the solution until the urea has dissolved Table 2 Preparation of a 5 Long Ranger Polyacrylamide Gel Component 5 Gel Final Concentration urea 18 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 4 Filter the acrylamide solution through a 0 2 micron filter e g Nalgene tissue culture filter and degas for 5 minutes 5 Add 35ul of TEMED and 250ul of fresh 10 ammonium persulfate to the 50ml of acrylamide solution and mix g2. 608 274 4330 Fax 608 277 2516 www promega com Part TBD021 Printed in USA Page 4 Revised 8 10 O 3 D Matrix Generation for the ABI PRISM 310 Genetic Analyzer 1 Open a new GeneMapper project To add matrix sample files to the new project select Add Samples to Project in the File menu Choose the appropriate run folder containing the fsa files from Section 3 C Highlight the run folder select Add To List then Add 2 To open the raw data for a specific matrix sample file locate Project in the upper left corner of the screen and double click on the run folder to reveal the fsa files 3 Choose a single fsa file to observe the raw data While viewing the raw data move the cursor to the region that is to the right of the primer peak and to the left of at least five peaks Choose a region in a flat part of the baseline 4 Record the data point value found at the lower left portion of the screen for use in Step 6 Repeat this step for each matrix standard Dye Color Corresponding Matrix Start At Value Blue Fluorescein Matrix Green JOE Matrix Yellow TMR Matrix Red CXR Matrix 5 To create a new matrix select GeneMapper Manager in the Tools menu Select the Matrices tab then New 6 Define the new matrix in the Matrix Editor Figure 1 Note The Matrix Name Start At values and Matrix Result values shown in Figure 1 will change depending on your instrument a Assign
3. Secs 3 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time minutes 30 Note The injection time may need to be increased or decreased depending on instrument sensitivity Peak heights of 1 000 4 000RFU are optimal for matrix generation 10 Select none for the matrix file 3 B Matrix Sample Preparation 1 Thaw the matrix standards For each matrix standard vortex the tube for 5 10 seconds to mix then add 2ul of matrix standard to 25ul of Hi Di formamide 2 Denature each sample for 3 minutes at 95 C and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading 3 Place 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 select Run to start the capillary electrophoresis system 2 Monitor the electrophoresis by observing the raw data and status windows Each sample will take approximately 40 minutes for syringe pumping sample injection and electrophoresis Note The matrix files that are created will be fsa files After the run is finished save or transfer the fsa files to a secure location where they can be opened in a GeneMapper project Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone
4. a matrix name in the Matrix Name field b Set Number of Dyes to 4 c To select each matrix standard sample file click on the dye color for each matrix B for fluorescein G for JOE Y for TMR and R for CXR Navigate to the fsa sample file that corresponds to that dye and double click on it to add the sample file Repeat this step for each matrix standard Note To find the fsa files in the default location go to My Computer AB SW8DATA D Applied Bio 310 then Runs and locate the correct run folder 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 TBD021 Revised 8 10 Page 5 F Matrix Editor gt Matrix Description Matrix Name POP4_4 dyeF_310PC Description Matrix Settings Select the Matrix Standard Sample File Number of Dyes W No File Selected for B Data Start At 1000 G No File Selected for G Data Stat At 1000 T No File Selected for Y Data Stat At 1000 No File Selected for R Data Start At 1000 100000 M Matrix Result oe 9106TA Figure 1 The Matrix Editor d Enter the data point value recorded from Step 4 in the Start at field Repeat this step for each matrix standard e Click on the Create button The Matrix Result should give a value of 1 000 when
5. ice water bath before loading the gel or capillary Denature samples just prior to loading Poor quality matrix CE related artifacts spikes Minor voltage extra peaks visible in changes or urea crystals passing by the laser can one or all color 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 Re inject the samples to confirm CE related artifacts contaminants Contaminants in the water used with the 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 prepare sample dilutions Change vials and wash the buffer reservoir 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 TBD021 Revised 8 10 Page 7 4 Troubleshooting continued Symptoms Causes and Comments Poor quality matrix elevated Matrix used was generated on another baseline and or inverted peaks instrument A matrix must be generated for in analyzed samples see Figure 2 each instrument Wrong dye was used Generate the matrix using the same dyes as those in the samples Oversubtraction of signal occurred because signal was saturated When generating a matrix avoid choosing
6. samples with peak heights that are higher than the recommended RFU values as this can result in a matrix that causes inverted peaks or elevated baseline Analyzed sample results may be improved by diluting the matrix samples in water before preparing them for use D S Cenescan Project 12 28 99 Display 1 SSS HH Elevated baseline 2883TA03_0A OW 0v 2008 Figure 2 Elevated baseline A sample was analyzed using an ABI PRISM 310 Genetic Analyzer and GeneScan analysis software The resulting electropherogram shows an elevated baseline below 270 bases 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 TBD021 Printed in USA Page 8 Revised 8 10 Symptoms Causes and Comments Inverted peaks in the matrix Incorrect or no Start At value was entered The baseline Figure 3 Start At value chosen in Section 3 D 5 A or 5 B should have a flat baseline Wrong colors were assigned to the dyes Confirm the dye and color selection Fluorescein Blue JOE Green TMR Yellow CXR Red Previously generated matrix Changes to or aging of instrument components no longer performs optimally Instrument sensitivity can change if the instrument was moved or recently serviced replacement or realignment of the laser CCD camera power supply or mirrors The sensitivity a
7. store a copy of the matrix file in the Matrix folder at C appliedbio shared analysis sizecaller matrix oO TMR matrix Ig mix 1 15 dil BC Io 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 2700 2400 2100 meena 900 300 g Start At aiei 3529 v 717 4 2882TA03_0A Figure 4 TMR matrix raw data The TMR matrix standard was analyzed using an ABI PRISM 310 Genetic Analyzer GeneScan analysis software was used to view the raw data in the Sample menu The cursor was placed on the baseline and the Start At value of 3529 was determined using the readout in the lower left corner of the window 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 TBD021 Printed in USA Page 16 Revised 8 10 O 6 A new matrix can be applied to previously run samples by highlighting the sample in the GeneScan project In the Sample menu select Install new matrix highlight the new matrix and select Open The new matrix will be applied to the sample file and the samples can be analyzed using the new matrix 5 C Composition of Buffers and Solutions 10 ammonium persulfate TBE 10X buffer Add 0 05g of ammonium persulfate 107 8g Tris base Cat V3131 to 500ul of deioniz
8. the ABI PRISM 310 Data Collection Software Version 3 1 0 2 To preheat the ABI PRISM 310 Genetic Analyzer to 60 C select Manual Control in the Window menu In the Function menu select Temperature Set Set Value to 60 0 then select Execute Close the Manual Control screen 3 Inthe File menu select New to open the Create New menu Open a GeneScan sample sheet either 48 Tube or 96 Tube 4 In the upper right corner of the sample sheet 4 Dyes should be selected Enter the appropriate sample information in the Sample Name field Matrix sample names should be descriptive for example add the color to the sample name Label tubes with the corresponding sample names 5 To save the sample sheet select Save As in the File menu Assign a name to the file and save to the Sample Sheet folder Close the file 6 In the File menu select New to open the Create New menu 7 Open the GeneScan injection list 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 TBD021 Revised 8 10 Page 3 3 A Instrument Preparation continued 8 Select the sample sheet i e the gss file that was created in Step 5 9 The recommended module is GS STR POP4 ImL F md4 choose this module using the pull down menu The settings should be Inj
9. Revised 8 10 Page 13 5 A Detection of Matrix Fragments Using the ABI PRISM 377 DNA Sequencer continued 4 Select OK and the matrix file will be generated 5 Save the matrix file in the Matrix Standards folder located in the GeneScan folder A copy of the matrix file should be stored in the ABI folder located in the system folder 6 Anew matrix can be applied to previously run samples by highlighting the sample in the GeneScan project In the Sample menu select Install new matrix highlight the new matrix and select Open The new matrix will be applied to the sample file and the samples can be analyzed using the new matrix Reuse of Glass Plates Separate the glass plates and discard the gel Clean glass plates with hot water and a detergent such as 1 Liqui Nox detergent Rinse extremely well with deionized water and allow plates to air dry Do not scrape plates with abrasive materials during this process Gel extrusion i e gel expands into the comb during a run can occur due to a buildup of residue If this occurs soak the plates in 2N HCI for 15 minutes then rinse thoroughly 5 B Detection of Matrix Fragments Using the ABI PRISM 310 Genetic Analyzer and GeneScan Software Materials to Be Supplied by the User e dry heating block water bath or thermal cycler e crushed ice or ice water bath e 310 capillaries 47cm x 504m e performance optimized polymer 4 POP 4 polymer e glass s
10. Technical Bulletin PowerPlex Matrix Standards 310 INSTRUCTIONS FOR USE OF PRODUCT DG4640 PRINTED IN USA Revised 8 10 Part TBD021 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 IPAR E IDR IM P AE ieee eee cess 1 2 Product Components and Storage Conditions oe eeeeteeeeteeneeneens 2 3 Detection of Matrix Fragments Using the ABI PRISM 310 Genetic Analyzer and GeneMapper ID Software cesses 3 Ae enen Tea TO ioatea naaa aiaa 3 B Matiy Sample Preparation gtsicasiatescrisciseoeenuerrbacctietinein nant avian 4 C Capillary Electrophoresis anid Detection sctinidiimcatbstiwainstinsantorsiveiatenousancatioed 4 D Matrix Generation for the ABI PRISM 310 Genetic Analyzer 5 d Troubleshoolin penssi aoaiina 7 She NA N Senet E ee ee 10 A Detection of Matrix Fragments Using the rN JG alta belt kaso Wie UNA Degu NCE soiis haa aaia 10 B Detection of Matrix Fragments Using the ABI PRISM 310 Genetic Analyzer and GeneScan Software s 14 C Composition ot Bu fters and SO WONG oiaiaccestesestate tiated seta ctsceeeerendeeeneeceon 17 0 Related Trod 1 Cc ee ce Pn Oven 17 1 Description Proper generation of a matrix file is critical to evaluate multicolor s
11. ach 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 gel temperature 8 Prepare the matrix standards during the gel prerun 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 TBD021 Printed in USA Page 12 Revised 8 10 Sample Preparation and Loading 1 Combine 1 5 of each matrix standard with 1 5ul of Blue Dextran Loading Solution 2 Denature each sample for 3 minutes at 95 C and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the gel Note Instrument detection limits vary therefore the amount of product mixed with loading cocktail may need to be increased or decreased 3 After the 15 to 20 minute prerun pause the instrument by selecting Pause When the prerun is paused the water will continue to circulate to keep the gel warm during the sample loading 4 Use a 30cc syringe filled with buffer to flush the urea from the well area 5 Load 1 5ul of each denatured sample into the respective wells 6 Place the lid on the upper buffer chamber and close the instrument door Gel Electrophoresis and Detection 1 After loading
12. comparing a dye to itself Typically all other values will be less than 1 000 Select OK and the matrix will be created in the Matrices tab of the GeneMapper Manager Select Done 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 TBD021 Printed in USA Page 6 Revised 8 10 4 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 Re inject the sample Check the syringe for leakage Check the laser power Poor quality formamide was used Use only fresh Hi Di formamide when running samples on the ABI PRISM 310 Genetic Analyzer Samples were degraded due to improper storage Store matrix standards in the dark at 20 C protected from light Do not store in the freezer door or in a frost free freezer Peak heights were 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 injection time or loading volume Samples were not denatured Heat denature samples and immediately chill on crushed ice or in an
13. cturer For information on other Promega fluorescent STR systems refer to the PowerPlex 16 System Technical Manual TMD012 PowerPlex 16 HS System Technical Manual TMD022 PowerPlex Y System Technical Manual TMD018 PowerPlex ES System Technical Manual TMDO017 PowerPlex S5 System Technical Manual TMD021 and GenePrint Fluorescent STR Systems Technical Manual TMD006 These Technical Manuals and additional product information are available at www promega com 2 Product Components and Storage Conditions Product Size Cat PowerPlex Matrix Standards 310 50ul each dye DG4640 Not for Medical Diagnostic Use Includes 50ul Fluorescein Matrix 50ul JOE Matrix 50ul TMR Matrix 50ul CXR Matrix Iml Blue Dextran Loading Solution Storage Conditions Store all components at 20 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 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 are available upon request from Promega or at www promega com Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA T
14. e registered trademarks 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 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 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 TBD021 Printed in USA Page 18 Revised 8 10
15. ed 7 44g EDTA water Use 250ul of 10 ammonium persulfate for each 50ml of acrylamide gel solution Blue Dextran Loading Solution 88 25 formamide 15mg ml blue dextran 41mM EDTA pH 8 0 Na EDTA 2H 0 55 0g boric acid Dissolve Tris base and EDTA in 800ml of deionized water Slowly add boric acid and monitor the pH until the desired pH of 8 3 is obtained Bring the volume to 1 liter with deionized water 6 Related Products Product Size Cat PowerPlex Matrix Standards 3100 3130 25ul each dye _ DG4650 PowerPlex 16 System 100 reactions DC6531 400 reactions DC6530 PowerPlex 16 HS System 100 reactions DC2101 400 reactions DC2100 PowerPlex Y System 50 reactions DC6761 200 reactions DC6760 PowerPlex S5 System 100 reactions DC6951 400 reactions DC6950 PowerPlex 1 2 System 100 reactions DC6101 PowerPlex ES System 100 reactions DC6731 400 reactions DC6730 Not for Medical Diagnostic Use Promega Corporation 2800 Woods Hollow Road Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 Printed in USA Revised 8 10 Madison WI 93711 5399 USA WWW promega com Part TBD021 6 Related Products continued Accessory Components Product Size Cat Internal Lane Standard 600 150ul DGI1071 Gold ST R 10X Buffer 1 2ml DM2411 Nuclease Free Water 50ml P1193 For Laboratory Use 2006 2010 Promega Corporation All Rights Reserved GenePrint and PowerPlex ar
16. ently 6 Using a disposable 60cc 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 maintaining a constant flow of solution gently tap the glass plates to assist the movement of solution to the bottom of the plates 7 Insert a 36 well sharkstooth comb or 34 well squaretooth comb between the glass plates Sharkstooth combs with 64 or 96 wells also can be used Note The gel can be stored overnight by placing a paper towel saturated with deionized water around the top and bottom and covering with plastic wrap to prevent the gel from drying out Crystallization of the urea will destroy the gel 8 Secure the comb with three evenly spaced clamps 9 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 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 TBD021 Revised 8 10 Page 11 5 A Detection of Matrix Fragments Using the ABI PRISM 377 DNA Sequencer continued Instrument Preparation 1 Open the ABI PRISM 377 Data Collection Software 2 Prepare a sample sheet as described in the GeneScan Analysis Softwa
17. hone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TBD021 Revised 8 10 Page 15 5 B Detection of Matrix Fragments Using the ABI PRISM 310 Genetic Analyzer and GeneScan Software continued Matrix Generation for the ABI PRISM 310 Genetic Analyzer 1 Open the GeneScan project 2 Review the raw data from the individual matrix standards 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 See Figure 4 Select an area for matrix generation For optimal results use as many peaks as possible 3 In the File menu select New then click on 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 Corresponding Matrix Start At Value Blue Fluorescein Matrix Green JOE Matrix Yellow TMR Matrix Red CXR Matrix 4 Select OK and the matrix file will be generated 5 Save the matrix file in the Matrix Standards folder located in the GeneScan folder 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
18. lso can change over time due to aging of the instrument These changes can result in poor matrix performance Generate a new matrix Oo GeneScan Project 2 14 00 Display 4 3300 3600 3900 4200 4500 4800 5100 5400 5700 6000 6300 6600 6900 7200 7500 7800 DE 208 JOE matrix Ig mix 1 15 dil DE 206 JOE matrix Ig mix 1 15 dil OW 207 JOE matrix Ig mix 1 15 dil BE 20R JOE matrix Ig mix 1 15 dil Te 2884TA03_0A Figure 3 Inverted baseline The four matrix samples from the PowerPlex Matrix Standards 310 were analyzed using 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 to the JOE Matrix sample file and analysis was performed using all four colors The result shows inverted peaks in the blue yellow and red channels 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 TBD021 Revised 8 10 Page 9 5 Appendix 5 A Detection of Matrix Fragments Using the ABI PRISM 377 DNA Sequencer Materials to Be Supplied by the User Solution compositions are provided in Section 5 C e dry heating block water bath or thermal cycler e crushed ice or ice water bath e Long Ranger gel solution Lonza Cat 50611 or Lo
19. ng Ranger Singel pack for ABI sequencers 377 36cm Lonza Cat 50691 e 10 ammonium persulfate TEMED e Urea Cat V3171 e 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 plates e 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 e 60cc syringe e 30cc syringe e 18 gauge needles O 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 gloves and safety glasses when working with acrylamide solutions 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 TEMED corrosive flammable urea irritant formamide irritant teratogen contained in the Blue Dextran Loading Solution Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
20. o column Create a new GeneScan injection list Select the appropriate sample sheet using the pull down menu 4 Select the GS STR POP4 1ml A Module using the pull down menu Change the run time to 30 minutes and keep the settings for the remaining parameters as shown below Inj Secs 3 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 Sample Preparation 1 Thaw the matrix standards For each matrix standard vortex the tube for 5 10 seconds to mix then add 2ul of matrix standard to 25ul of Hi Di formamide or water 2 Denature each sample for 3 minutes at 95 C and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading 3 Place tubes in the appropriate autosampler tray 48 tube or 96 tube 4 Place the autosampler tray in the instrument and close the instrument doors Capillary Electrophoresis and Detection 1 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system 2 Monitor the electrophoresis by observing the raw data and status windows Each sample will take approximately 40 minutes for syringe pumping sample injection and electrophoresis Promega Corporation gt 2800 Woods Hollow Road gt Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 P
21. oll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TBD021 Printed in USA Page 2 Revised 8 10 3 Detection of Matrix Fragments Using the ABI PRISM 310 Genetic Analyzer and GeneMapper ID Software Materials to Be Supplied by the User e dry heating block water bath or thermal cycler e crushed ice or ice water bath e 310 capillaries 47cm x 50um e performance optimized polymer 4 POP 4 polymer e 10X genetic analyzer buffer e sample tubes and septa e aerosol resistant pipette tips 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 3 A Instrument Preparation 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 1 Open
22. re User s Manual Enter the appropriate sample information in the Sample Info column 3 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 4 Select the appropriate sample sheet and comb selection using the pull down menus 5 Select none for the gel matrix file Gel Prerun 1 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 2 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 3 Position the gel glass plate unit in the 377 cassette 4 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 5 Add 1X TBE buffer to the top and bottom buffer chambers of the instrument 6 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 18 gauge needle remove any air bubbles from the bottom of the gel 7 Att
23. select Cancel to stop the prerun Make sure that the run time is set at 3 hours then select Run to begin electrophoresis 2 Monitor electrophoresis by observing the gel image and status windows 3 Allow electrophoresis to proceed for 3 hours The largest fragment will have migrated past the laser 4 Track and extract the gel lanes Matrix Generation for the ABI PRISM 377 DNA Sequencer 1 Open the GeneScan project 2 Review the raw data from the individual matrix samples 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 for matrix generation For optimal results use as many peaks as possible See Figure 4 3 In the File menu select New then click on 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 Start At Value Blue Fluorescein Matrix Green JOE Matrix Yellow TMR Matrix Red CXR Matrix 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 TBD021
24. yringe 1ml e sample tubes and septa e aerosol resistant pipette tips e 10X genetic analyzer buffer Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze Q 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 Q 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 Part TBD021 Printed in USA Page 14 Revised 8 10 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 Inf
25. ystems with the ABI PRISM 310 Genetic Analyzer and ABI PRISM 377 DNA Sequencer To prepare a matrix four standards are analyzed using the same electrophoresis conditions as those for samples and allelic ladders The PowerPlex Matrix Standards 310 consists of DNA fragments labeled with four fluorescent dyes one tube contains DNA fragments labeled with fluorescein one tube contains DNA fragments labeled with carboxy tetramethylrhodamine TMR one tube contains DNA fragments labeled with 6 carboxy 4 7 5 dichloro 27 7 dimethoxyfluorescein JOE and one tube contains DNA fragments labeled with carboxy X rhodamine CXR 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 TBD021 Revised 8 10 Page 1 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 can be used with the PowerPlex 16 PowerPlex 16 HS PowerPlex Y PowerPlex ES and PowerPlex S5 Systems It also can be used with the PowerPlex 1 2 System fluorescein and TMR labeled or any of the fluorescent STR systems fluorescein labeled A matrix should be generated for each individual instrument Protocols to operate the fluorescence detection instrumentation should be obtained from the manufa
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