Manual - Thermo Fisher Scientific
Contents
1. Protocols for Processing AmpFL STR PCR Amplification Kit Products 17 18 Verifying the To verify the matrix file Matrix File l Select File gt Open gt Matrix and verify that the values on the diagonal from top left to bottom right are 1 000 Reactions B G M R D 8 ME oas oos foooa2 o0052 G fosssa 10000 fozses foores oo055 Y fosios fo71s9 10000 fosss0 fooos2 R fo221e fossie fossss 1 0000 o 0040 o foo221 fooaze foosss fo1s31 10000 Note The values obtained are unique for each instrument for each virtual filter set and for each specific set of run conditions Apply the new matrix file to the Matrix Standard Sample Files a Inthe Analysis Control window highlight the Sample File column by clicking in the Sample File title row b Select Sample gt Install New Matrix c Select the new matrix file located in the Matrix folder D AppliedBio Shared Analysis SizeCaller Matrix d Click Open Analyze all of the matrix standard samples a Select Settings gt Analysis Parameters and verify that the settings are correct b In the Analysis Control window select For Dye Set 33 All five colors B G Y R and O For Dye Set 32 Four colors B G Y and R c Click Analyze Protocols for Processing AmpFLSTR9 PCR Amplification Kit Products Matrix File Creation To verify the matrix file continued 4 In the Results2. n RR 7200 5400 3600 1800 E Gl Y ALIS Y xj gt Figure 1 GeneScan 500 LIZ Size Standard peaks Note In Figure 1 the 450 bp peak is assigned when using the GeneScan 500 LIZ Size Standard When using the GeneScan 500 ROX Size Standard figure not shown do not assign the 450 bp peak Protocols for Processing AmpFLSTR PCR Amplification Kit Products 29 30 To analyze data with GeneScan software continued 3 Save the size standard in the SizeStandards folder D AppliedBio Shared Analysis SizeCaller SizeStandards To apply the size standard to all injections select the appropriate standard in the Size Standard column header above sample 1 in the Analysis Control window Analyze sample files a Highlight the appropriate columns For Dye Set 33 Blue green yellow red and orange For Dye Set 32 Blue green yellow and red b Confirm that a diamond symbol appears in all appropriate boxes where a size standard is included with the sample files For five dye samples Confirm that the orange box is the standard For four dye samples Confirm that the red box is the standard Note If the diamond symbol does not appear in the appropriate boxes Ctrl click to place a diamond in the box c Click Analyze Protocols for Processing AmpFLSTR9 PCR Amplification Kit Products Data Analysis To analyze data with GeneScan software continued
3. Equipment Source Gloves disposable powder free MLS Ice bucket MLS Lab coat MLS Lint free tissues MLS Microcentrifuge tubes 1 5 mL MLS Microtube racks MLS Nalgene filter apparatus 150 mL 0 2 um CN MLS filter Permanent ink pen MLS Pipet bulb MLS Pipet tips sterile disposable hydrophobic filter MLS plugged Pipets serological MLS Pipettors adjustable 1 10 uL 2 20 uL MLS 20 200 uL and 200 1000 uL Refrigerator MLS Repeat pipettor and Combitips that dispense MLS 25 125 uL optional Syringe 35 cc optional MLS Tape MLS Tube 50 mL Falcon MLS Tube decapper autoclavable MLS Protocols for Processing AmpFLSTR PCR Amplification Kit Products 5 Reagents Table3 Reagents Reagent Source ABI PRISM 310 10X Genetic Analyzer Buffer with EDTA Applied Biosystems PN 402824 AmpF STR Blue PCR Amplification Kit Applied Biosystems PN 402800 AmpF STR COfiler PCR Amplification Kit Applied Biosystems PN 4305246 AmpF STR Green I PCR Amplification Kit Applied Biosystems PN 4307133 AmpF STR Identifiler PCR Amplification Kit Applied Biosystems PN 4322288 AmpF STR Profiler Plus and AmpF STR COfiler PCR Amplification Kits Applied Biosystems PN 4305979 AmpF STR Profiler Plus ID PCR Amplification Kit Applied Biosystems PN 4330284 AmpF STR Profiler Plus PCR Amplification Kit Applied Biosystems PN 4303326
4. 6 After the analysis is complete confirm that the sizes for the size standard peaks are correctly assigned a Select Window gt Results Control and examine the appropriate size standard peaks in overlapping groups of 16 samples Quick Tile Off For five dye samples Examine the orange GeneScan 500 LIZ Size Standard peaks For four dye samples Examine the red GeneScan 500 ROX Size Standard peaks Note Be sure to select View gt Align By Size b While the samples are tiled check the 250 bp peaks sized as approximately 246 bp in the enlarged view window Remember that this peak was not defined in the size standard The tiled 250 bp peaks should size consistently and should all overlap In a typical run the 250 bp peaks all fall within a size window of approximately 1 bp Note Laboratory temperature variations can cause size variations 71 bp If the temperature of the laboratory varies try injecting the AmpFZSTR Allelic Ladder approximately every 10 injections or 5 hours c Scroll through the tables to verify correct size standard peak assignments d Check the size standard peaks in the remaining samples taking note of which samples if any have incorrect peak assignments Displaying For more information about displaying the results refer to the Results 4BI PnisM GeneScan Analysis Software Version 3 7 User S Manual PN 4308923 Analyzing Data For more information about analyzing data with G
5. preparation Sample Sheet of samples and saved in the Sample Sheet folder and Injection List To create a sample sheet and injection list l Using 310 Data Collection Software v3 0 select File New and click the appropriate GeneScan Sample Sheet icon Create new x a f amp j tuj oe Sequence GeneScan Sequence Sequence GeneScan GeneScan Injection Injection Smpl Sheet Smpl Sheet Smpl Sheet Smpl Sheet List List 48 Tube 96 Tube 48 Tube 96 Tube Make a selection from the drop down menu For five dye samples 5 dyes For four dye samples 4 dyes Enter sample names and numbers for each injection in the Sample Name column This column indicates which sample is in which tube of the sample tray Protocols for Processing AmpFL STR PCR Amplification Kit Products Instrument Setup To create a sample sheet and injection list continued 4 For each sample enter the sample description for each row in the Sample Info column For five dye samples Blue Green Yellow and Red For four dye samples Blue Green and Yellow Note Sample descriptions are necessary for the AmpF STR template file to build tables containing the genotypes for each sample Note If you are using Profiler Plus and COfiler PCR Amplification Kits together with Genotyper Software v3 7 and the combined COPP COfiler and Profiler Plus template files enter the information as follows Amp
6. sheet and injection list continued 9 Select the module file for every injection a Click the arrow in the Module column for the first sample injection to view the pop up menu and select For Dye Set 33 GS STR POP4 1 mL G5v2 module file For Dye Set 32 GS STR POP4 1 mL F module file b Select the entire Module column by clicking the Module column heading then select Edit Fill Down Note You do not need to perform this step if the preferences were set as described in Setting the Data Collection Software Preferences on page 20 Select matrix files for the injections a From the Matrix file pop up menu select the appropriate matrix file for each injection b Click the arrow in the Matrix column for the first sample injection to view the pop up menu and select the appropriate matrix file Select the entire Matrix column by clicking the Matrix column heading then select Edit gt Fill Down IMPORTANT The matrix file must be one that was made using the appropriate matrix standards and filter set module To autoanalyze place a copy of the matrix file in the Matrix folder D AppliedBio Shared Analysis SizeCaller Matrix Note You do not need to perform this step if the preferences were set as described in Setting the Data Collection Software Preferences on page 20 Protocols for Processing AmpFL STR PCR Amplification Kit Products 25 Electrophoresis Procedural Overvi
7. Aliquot the Hi Di Formamide size standard mixture a Label 0 2 mL or 0 5 mL Genetic Analyzer sample tubes appropriately b Aliquot 25 uL of the Hi Di Formamide size standard mixture into the labeled tubes Note To pipet the Hi Di Formamide size standard mixture Applied Biosystems recommends using a repeating pipettor Add 1 5 uL of PCR product or AmpFZSTR Allelic Ladder per tube and mix by pipetting up and down Seal each tube with a septum Vortex the sample tray and then centrifuge the tray briefly Note Make sure that there are no bubbles Denature each sample for 3 minutes at 95 C Chill tubes for at least 3 minutes on ice Note Be careful not to carry over any water on the outside of the tubes Water on the autosampler tray may promote arcing Loading and To load and run samples Running Samples l Open the instrument doors and press the Tray button to present the autosampler Place a 48 well or 96 well sample tray on the autosampler Note For a 48 well autosampler tray place tube 1 into sample tray position A1 tube 2 into sample tray position A3 and so on Note For a 96 well autosampler tray place tube 1 into sample tray position A1 tube 2 into sample tray position A2 and so on Protocols for Processing AmpF STR PCR Amplification Kit Products 27 28 To load and run samples continued 3 Press the Tray button on the inst
8. AmpF STR SEfiler PCR Amplification Kit Applied Biosystems PN 4335129 AmpF STR SGM Plus PCR Amplification Kit Applied Biosystems PN 4307133 GeneScan 500 LIZ Size Standard Applied Biosystems PN 4322682 GeneScan 500 ROX Size Standard Applied Biosystems PN 401734 Hi Di Formamide Applied Biosystems PN 4311320 Matrix Standard Set DS 32 5 FAM JOE NED ROX dyes for use with the 310 377 system Applied Biosystems PN 4312131 Matrix Standard Set DS 33 6 FAM VIC NED PET LIZ dyes for use with the 310 377 system Applied Biosystems PN 4318159 Protocols for Processing AmpFLSTR9 PCR Amplification Kit Products Dedicated Equipment and Supplies Table 3 Reagents continued Reagent Source POP 4 Performance Optimized Polymer Applied Biosystems PN 402838 Deionized water PCR grade MLS Documents Table 4 Applied Biosystems documents Document Part Number ABI PRISM 310 Genetic Analyzer User Bulletin 4339367 G5v2 Module for Use with Dye Set 33 DS 33 ABI PRISM 310 Genetic Analyzer User Guide 4317588 ABI PRISM GeneScan Analysis Software for the 4335617 Windows NT Operating System User Bulletin Overview of the Analysis Parameters and Size Caller ABI PRISM GeneScan Analysis Software 4308923 Version 3 7 User s Manual ABI Prism Genotyper 3 7 NT Software User s 4309947 Ma
9. Control window examine the results for all colors for each of the matrix standard samples na five dye system the 6 FAM matrix standard results should have peaks for blue Evaluate the baseline A pattern of pronounced peaks or dips in any of the other four colors indicates that the color separation may not be optimal na four dye system the 5 FAM matrix standard results should have peaks for blue Evaluate the baseline A pattern of pronounced peaks or dips in any of the other three colors indicates that the color separation may not be optimal Protocols for Processing AmpFLSTR PCR Amplification Kit Products 19 Instrument Setup Procedural Overview Setting the Data Collection Software Preferences 20 Instrument setup involves l 2 3 Setting the data collection software preferences Setting the run temperature Creating a sample sheet and injection list To set data collection software preferences l Start the 310 Data Collection Software v3 0 2 Select Window gt Preferences Note This is an optional step Set the standard color a Select the GeneScan Sample Sheet Defaults tab b Set the Size Standard Dye Color 5 Dye to O Orange c Setthe Size Standard Dye Color 4 Dye to R Red Size Standard Dye Color 4 Dye R bd Size Standard Dye Color 5 Dye o M Protocols for Processing AmpF STR PCR Amplification Kit Products Instrument Setup To set dat
10. F STR Kit Used Sample Info Comments Profiler Plus PCR Unique sample Profiler Plus Amplification Kit description COfiler PCR COfiler Amplification Kit For each sample enter the same Sample Info for both kits used Protocols for Processing AmpFLSTR PCR Amplification Kit Products 23 To create a sample sheet and injection list continued 5 For each AmpF STR Allelic Ladder injection type ladder in the Sample Info column For five dye samples Blue Green Yellow and Red For four dye samples Blue Green and Yellow Note The Genotyper software requires the word ladder Note If you are using Profiler Plus and COfiler PCR Amplification Kits together with Genotyper Software v3 7 and the combined COPP COfiler and Profiler Plus template files enter the information as follows AmpF STR Allelic a Ladder Used Sample Info Comments Profiler Plus ladder PPL COfiler COL Entries are case sensitive Make sure that the diamond symbol in the Std column indicates the appropriate standard For five dye samples Orange For four dye samples Red Select File gt New and click the GeneScan Injection List icon Select the appropriate sample sheet from the Sample Sheet pop up menu at the top left of the Injection List window Protocols for Processing AmpFLSTR9 PCR Amplification Kit Products Instrument Setup To create a sample
11. TR9 PCR Amplification Kit Products Safety Chemical Waste Neem CHEMICAL WASTE HAZARD Some wastes Hazard Warning produced by the operation of the instrument or system are potentially hazardous and can cause injury illness or death Chemical Waste To minimize the hazards of chemical waste Guidelines Read and understand the MSDSs for the chemicals in a waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks that may occur from the primary container Both must be compatible with the waste material and meet national state provincial and local requirements for container storage Minimize contact with and inhalation of chemical waste When handling chemicals wear appropriate personal protective equipment such as safety glasses gloves and protective clothing Handle chemical wastes in a fume hood After you empty a chemical waste container seal it with the cap provided Dispose of the contents of a waste container in accordance with good laboratory practices and local state provincial and or national environmental and health regulations Site Preparation A site preparation and safety guide is a separate document sent to all and Safety Guide customers who have purchased an Applied Biosystems instrument Refer to the guide written for your instrument for inform
12. User Bulletin ABI PRISM 310 Genetic Analyzer SUBJECT In This User Bulletin February 7 2003 Protocols for Processing AmpF STR PCR Amplification Kit Products with the Microsoft Windows NT Operating System Dedicated Equipment and Supplies 00000 eaee 4 Safety cee ewe ene ered T RH ee Keka Sheswie Mikes Mes es 9 Matrix File Creation 00 0 cc ccc eee ene 13 Instrument Setup 2 0 eee eee 20 Electrophoresis caror rinri rimeno Ab OS Oe EE aedes 26 Data Analysis 53h bb Gi eee di dinar di Re AE eR RR RUE 29 Before You Begin Applicable AmpF STR Kits Software Requirements PCR products generated from any of the AmpF STR kits may be used with the protocols described in this user bulletin The protocols in this user bulletin require the following software and files to be installed on your computer Microsoft Windows NT operating system 310 Data Collection Software v3 0 PN 4326986 ABI PRISM GeneScan Analysis Software v3 7 1 ABI PRISM Genotyper Software v3 7 Appropriate run module file GS STR POP4 1 mL G5v2 module file or GS STR POP4 1 mL G5 module file GS STR POP4 1 mL F module file Note For more information about the G5v2 module see the ABI PRISM 310 Genetic Analyzer User Bulletin G5v2 Module For Use with Dye Set 33 DS 33 PN 4339367 Applied Biosystems Product Usage Use the software products as shown in Table 1 Note Wherever G5v2 app
13. a collection software preferences continued 4 Select the GeneScan Injection List Defaults tab and make the following selections a Select the appropriate module 5 Dye Module GS STR POP4 1 mL G5v2 4 Dye Module GS STR POP4 1 mL F b Select an appropriate matrix file c Select the Autoanalyze with file If you plan to autoanalyze select AnalyzeGSSample bat If you do not plan to autoanalyze your data select lt none gt Note When you create a new sample sheet a portion of the form is automatically completed for you You can modify the automatic defaults in the Preferences file Click OK to save your changes Setting the Run Setting the run temperature prior to starting a run is optional Temperature however this step saves time This heating step occurs automatically at the beginning of the run modules To set the run temperature l Close the instrument doors 2 Return to the 310 Data Collection Software v3 0 Set the temperature a Select Window Manual Control b Select Temperature Set from the pop up menu c Set the temperature to 60 C d Click Execute Note It takes up to 30 min for the instrument to reach the 60 C run temperature You can prepare samples while the instrument is heating Protocols for Processing AmpFLSTR9 PCR Amplification Kit Products 21 22 Creating a The sample sheet can be prepared at any time before the
14. and female reproductive systems and is a possible birth defect hazard Please read the MSDS and follow the handling instructions Wear appropriate protective eye wear clothing and gloves IMPORTANT Do not include the size standard in the preparation of the matrix standards Heat the tubes at 95 C for 3 minutes to denature the DNA Immediately place the tube on ice for 3 minutes Place tubes in the sample tray IMPORTANT Make sure that you do not carry over any water on the outside of the tubes Water on the autosampler tray may promote arcing Protocols for Processing AmpFL STR PCR Amplification Kit Products 15 Performing a_ After you create a satisfactory matrix file you can apply this matrix Matrix File Run file to subsequent runs It is not necessary to run matrix standard samples for each new capillary To perform a matrix file run l Start the 310 Data Collection Software v3 0 2 Create a sample sheet for the matrix standard samples a Select File gt New and click the appropriate GeneScan sample sheet icon b Enter the sample names numbers for each row in the Sample Name column to identify which sample is in which tube of the sample tray c Close and save the sample sheet Note For more information refer to the ABI PRISM 310 Genetic Analyzer User Guide PN 4317588 Create an injection list for each matrix standard sample a Select File gt New and cli
15. ation on site preparation instrument safety chemical safety and waste profiles Waste profiles help you plan for the handling and disposal of waste generated by operation of the instrument Read the waste profiles and all applicable MSDSs for your instrument before handling or disposing of chemical waste Protocols for Processing AmpFLSTR PCR Amplification Kit Products 11 Waste Disposal Biological Hazard Safety 12 If potentially hazardous waste is generated when you operate the instrument e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste 1s stored transferred transported and disposed of according to all local state provincial and or national regulations Note Radioactive or biohazardous materials may require special handling and disposal limitations may apply ANCE BIOHAZARD Biological samples such as tissues body fluids and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves Read and follow the guidelines in these publications US Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Labora
16. ck the GeneScan Injection List icon b In the Injection List select the appropriate sample sheet from the Sample Sheet pop up menu c For every injection select Module gt GS STR POP4 1 mL G5v2 for Dye Set 33 Module gt GS STR POP4 1 mL F for Dye Set 32 d In the Matrix File column select None Click Run 16 Protocols for Processing AnpFLSTR PCR Amplification Kit Products Matrix File Creation Creating the When the injections are completed create the matrix using the Matrix File GeneScan Analysis Software To create the matrix file 1 Start the GeneScan Analysis Software v3 7 1 2 Select File gt New 3 Click the Matrix icon and select from the number of dyes pop up window Five dyes for Dye Set 33 Four dyes for Dye Set 32 4 In the window that opens a Indicate the sample files that correspond to each matrix standard dye color b Select starting scan numbers for each sample to exclude the primer peak c Select the number of points so that the matrix standard peaks are contained in the scanned region approximately 2500 scan data points Avoid spikes or artifacts 1f possible when selecting the range Note Review data of each matrix standard Reinject if necessary 5 Click OK to create the matrix and open the matrix file table 6 Save the matrix file in the Matrix folder D AppliedBio Shared Analysis SizeCaller Matrix
17. d can result in serious injury or death N DIA elss Indicates an imminently hazardous situation that 1f not avoided will result in serious injury or death This signal word Is to be limited to the most extreme situations Chemical Hazard erm CHEMICAL HAZARD Some of the chemicals Warning used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Chemical Safety To minimize the hazards of chemicals Guidelines Read and understand the MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs Minimize contact with chemicals When handling chemicals wear appropriate personal protective equipment such as safety glasses gloves and protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example a fume hood For additional safety guidelines consult the MSDS Protocols for Processing AmpFLSTR PCR Amplification Kit Products 9 About MSDSs Obtaining MSDSs 10 Check regularly for chemical leaks or spills If a leak or spill occurs follow the cleanup procedures recommended in the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Chemical manufactu
18. ds for AmpFZSTR kits Kit Dye Set Run Module Matrix Standard e AmpF STR Identifiler Dye Set 33 Filter Set G5v2 Matrix Standard Set DS 33 PCR Amplification Kit e 6 FAM dye PN 4318159 e AmpF STR SEfiler PCR Amplification Kit VIC dye e NED dye e PET dye e LIZ dye e AmpF STR COfiler Dye Set 32 Filter Set F Matrix Standard Set DS 32 dc sees Kit 5 FAM dye PN 4312131 e Amp rofiler m PCR Amplification Kit JOE dye e NED dye e AmpF STR Profiler sA Plus PCR Amplification ROX dye Kit e AmpF STR Profiler Plus D PCR Amplification Kit e AmpF STR SGM Plus PCR Amplification Kit e AmpF STR Blue PCR Amplification Kit e AmpF STR Green I PCR Amplification Kit 14 Protocols for Processing AnpFLSTR PCR Amplification Kit Products Matrix File Creation Preparing the To prepare the matrix standards Matrix Standards l Vortex the matrix standard set tubes thoroughly and then centrifuge them briefly For Dye Set 33 Use the Matrix Standard Set DS 33 For Dye Set 32 Use the Matrix Standard Set DS 32 Prepare one tube for each matrix standard sample by combining in each tube Matrix standard 1 uL Hi Di Formamide 24 uL erm CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and may cause irritation to the eyes skin and respiratory tract It may cause damage to the central nervous system and the male
19. ears in this document both G5v2 and G5 apply Table1 Product usage Product Usage 310 Data Collection Software v3 0 e Running AmpF STR PCR Amplification Kit products e Collecting five dye and four dye data Filter Set G5v2 module files Running samples controls and matrix standards Matrix Standard Set DS 33 6 FAM VIC NED PET and LIZ dyes for the 310 377 systems using the GS STR POP4 1 mL G5v2 module Creating the required matrix file for Dye Set 33 DS 33 Filter Set F module files Running samples controls and matrix standards Matrix Standard Set DS 32 6 FAM JOE NED and ROX dyes for the 310 377 systems using the GS STR POP4 1 mL F module Creating the required matrix file for Dye Set 32 DS 32 ABI PRISM GeneScan Analysis Software for the Windows NT operating system v3 7 1 Detecting peaks and sizing data ABI PRISM Genotyper Software v3 7 Analyzing and genotyping kit data 2 Protocols for Processing AmpFLSTR9 PCR Amplification Kit Products Filter Set G5v2 Module Files Requirements Filter Set F Module Files Requirements Protocols for Processing AmpFLSTR PCR Amplification Kit Products Before You Begin The GS STR POP4 1 mL G5v2 module file must be used for Dye Set 33 DS 33 6 FAM VIC NED PET and LIZ dyes on the ABI PRISM 310 Genetic Analyzer Note Wherever Filter Set G5v2 appears in this document bo
20. enotyper software with Genotyper refer to the ABI PRISM Genotyper 3 7 NT Software User s Manual Software PN 4309947 Protocols for Processing AmpFLSTR PCR Amplification Kit Products 31 Copyright 2002 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures For AmpF STR PCR Amplification Kits For Research Forensic or Paternity Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document TRADEMARKS ABI Prism AmpF STR Applied Biosystems COfiler GeneScan Genotyper Identifiler LIZ Profiler Profiler Plus SGM Plus and VIC are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries AB Design Applera FAM Hi Di JOE NED PET POP 4 ROX and SEfiler are trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries GeneAmp is a registered trademark of Roche Molecular Systems Inc Microsoft and Windows NT are registered trademarks of Microsoft Corporation All other trademarks are the so
21. ew Preparing Samples and AmpF STR Allelic Ladder 26 Electrophoresis involves l Preparing samples and AmpF STR Allelic Ladder 2 Loading and running samples To prepare the samples l For the master mix combine the necessary amount of Hi Di Formamide and size standard in a single microcentrifuge tube IMPORTANT Be sure to include at least one injection of AmpF STR Allelic Ladder per run in the calculations For five dye samples Number of samples 2 x 24 5 uL Hi Di Formamide Number of samples 2 x 0 5 uL GeneScan 500 LIZ Size Standard For four dye samples Number of samples 2 x 24 uL Hi Di Formamide Number of samples 2 x 1 uL GeneScan 500 ROX Size Standard Note If you are using a multi channel pipettor or processing many samples you may need to prepare additional master mix wer CHEMICAL HAZARD Formamide is harmful if absorbed through the skin and may cause irritation to the eyes skin and respiratory tract It may cause damage to the central nervous system and the male and female reproductive systems and is a possible birth defect hazard Read the MSDS and follow the handling instructions Wear appropriate protective eye wear clothing and gloves Vortex the tube to mix and then centrifuge it briefly Protocols for Processing AmpFLSTR9 PCR Amplification Kit Products Electrophoresis To prepare the samples continued 3
22. le property of their respective owners Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com www appliedbiosystems com Applied Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 02 2003 Part Number 4341742 Rev A Stock Number 112UB04 01 an Applera business
23. nt The equipment and supplies necessary or recommended for running AmpF STR kit data on the ABI PRISM 310 Genetic Analyzer are listed in the tables below Unless otherwise noted many of the items listed are available from major laboratory suppliers MLS Note Amplified DNA equipment and supplies used to handle amplified DNA should not be taken out of the amplified DNA work area Samples that have not yet been amplified should never come into contact with these supplies and equipment Table 2 Equipment GeneAmp PCR System 9700 Equipment Source Instruments ABI PRISM 310 Genetic Analyzer See your Applied Biosystems sales representative ABI PRISM 310 Genetic Analyzer Accessories 1 0 mL Glass Syringe Applied Biosystems PN 4304471 310 Capillaries 47 cm x 50 um i d internally uncoated Applied Biosystems PN 402839 Genetic Analyzer Buffer Vials 4 0 mL Applied Biosystems PN 401955 Genetic Analyzer Sample Tubes 0 5 mL Applied Biosystems PN 401957 Genetic Analyzer Septa for 0 5 mL Sample Tubes Applied Biosystems PN 401956 Miscellaneous Equipment Benchkote absorbent protector sheets MLS Freezer 15 to 25 C non frost free MLS Glassware MLS Protocols for Processing AnpFLSTR PCR Amplification Kit Products Dedicated Equipment and Supplies Table 2 Equipment continued
24. nual AmpFLSTR Blue PCR Amplification Kit User s 402827 Manual AmpFtSTR Green I PCR Amplification Kit 402944 User s Manual AmpFLSTR COfiler PCR Amplification Kit 4305469 User s Manual AmpFt STR Igentifiler PCR Amplification Kit 4323291 User s Manual AmpFLSTR Profiler Plus ID PCR Amplification 4330429 Kit User Bulletin AmpFt STR Profiler Plus PCR Amplification Kit 4303501 User s Manual Protocols for Processing AmpFLSTR PCR Amplification Kit Products 7 Table 4 Applied Biosystems documents continued Document Part Number AmpFtSTR Profiler PCR Amplification Kit 402945 User s Manual AmpFt STR SEfiler PCR Amplification Kit 4337410 User s Manual AmpFt STR SGM Plus PCR Amplification Kit 4309589 User s Manual Protocols for Processing AmpFLSTR9 PCR Amplification Kit Products Safety Safety Safety Alert Four safety alert words appear in Applied Biosystems user Words documentation Each word implies a particular level of observation or action as described below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical N ize Indicates a potentially hazardous situation that if not avoided can result in minor or moderate injury It can also alert against unsafe practices damage to an instrument or loss of data N Mel Indicates a potentially hazardous situation that if not avoide
25. rers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files You can obtain from Applied Biosystems the MSDS for any chemical supplied by Applied Biosystems This service is free and available 24 hours a day To obtain MSDSs 1 Go to https docs appliedbiosystems com msdssearch html 2 Inthe Search field type in the chemical name part number or other information that appears in the MSDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose 4 To have a copy of a document sent by fax or e mail select Fax or Email to the left of the document title in the Search Results page then click RETRIEVE DOCUMENTS at the end of the document list 5 After you enter the required information click View Deliver Selected Documents Now Protocols for Processing AmpFLS
26. rument to retract the autosampler 4 Close the instrument doors 5 Click Run Note If you have not preheated the heat plate the module has an initial step in which the plate is heated to 60 C before running the first sample This step takes up to 30 min After the plate reaches 60 C the run will begin Protocols for Processing AmpFLSTR9 PCR Amplification Kit Products Data Analysis Data Analysis Procedural Overview Setting Analysis Preferences Analyzing Data with GeneScan Software Data analysis involves 1 Setting analysis preferences 2 Analyzing data with GeneScan software 3 Displaying results 4 Analyzing data with Genotyper software For more information about the analysis parameters refer to the ABI PRISM GeneScan Analysis Software Version 3 7 User Manual PN 4308923 and the ABI PRISM GeneScan Analysis Software for the Windows NT Operating System User Bulletin Overview of the Analysis Parameters and Size Caller PN 4335617 To analyze data with GeneScan software 1 Start the GeneScan Analysis Software v3 7 1 2 Ctrl click the arrow in the Size Standard column for a sample file to view the pop up menu and select New Note Do not assign a size for the 250 bp peak for data generated on the 310 instrument Skip a row or assign a size of zero This peak can be used as an indicator of precision within a run 3500 4000 4500 5000 5500 6000 6500 mM 1
27. th Filter Set G5v2 and Filter Set G5 apply Processing AmpF STR PCR Amplification Kit products that use 6 FAM VIC NED PET and LIZ dyes requires 1 Installing the Filter Set G5v2 module file on the instrument computer if not already present 2 Creating a matrix file using the Matrix Standard Set DS 33 6 FAM VIC NED PET and LIZ dyes for the 310 377 system using the GS STR POP4 1 mL G5v2 module 3 Running samples on the 310 instrument using Filter Set G5v2 4 Analyzing samples with the matrix file created using Filter Set G5v2 IMPORTANT Do not apply the G5v2 matrix file to data analyzed using the G5 module A matrix file produced using the G5v2 module does not use the same virtual filter settings as a matrix file produced using the G5 module When using the G5v2 module to collect sample data use a matrix file created using the G5v2 module The GS STR POP4 1 mL F module file must be used for Dye Set 32 DS 32 5 FAM JOE NED and ROX dyes on the ABI PRISM 310 Genetic Analyzer Processing AmpF STR PCR Amplification Kit products that use 5 FAM JOE NED and ROX dyes requires 1 Creating a matrix file using the Matrix Standard Set DS 32 5 FAM JOE NED and ROX dyes for the 310 377 system using the GS STR POP4 1 mL F module 2 Running samples on the 310 instrument using Filter Set F 3 Analyzing samples with the matrix file created using Filter Set F Dedicated Equipment and Supplies Equipme
28. tories stock no 017 040 00547 4 http bmbl od nih gov Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html Additional information about biohazard guidelines is available at http www cdc gov Protocols for Processing AmpFLSTR9 PCR Amplification Kit Products Matrix File Creation Matrix File Creation Purpose The GeneScan Analysis software requires the matrix file to address spectral overlap of the dyes when performing multicomponent analysis of samples with multiple markers and colors in one injection When to Prepare Prepare a new matrix file Matrix Files Foreach instrument Fora particular set of run conditions Atleast one time each month to use with AmpFZSTR products After changing the lot of polymer capillaries and or buffer After service of the optics or other associated hardware Procedural Matrix file creation involves Overview 1 Choosing the dye set run module and matrix standard Preparing the matrix standards Performing a matrix file run Creating the matrix file Sn Aa at Verifying the matrix file Protocols for Processing AmpFLSTR PCR Amplification Kit Products 13 Choosing the Choose the appropriate dye set run module and matrix standard for Dye Set Run the AmpF STR kit you are using as shown in Table 5 Module and Matrix Standard Table 5 Dye sets run modules and matrix standar
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