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PowerPlex® 16 System

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1. 31 F Sample Analysis Using the Genotyper Software and PowerTyper 16 Macro 32 G Controls 35 H Results 35 VII Troubleshooting 37 A Amplification and Fragment Detection 37 B GeneMapper ID Analysis Software 40 C PowerTyper 16 Macro 43 VIII References 44 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 TMD012 Revised 5 08 PowerPlex 16 System All technical literature is available on the Internet at www promega com tbs Please visit the web site to verify that you are usin
2. Apply the stored analysis parameters file to the samples 6 Assign a new size standard Select a sample file highlight the arrow next to size standard then select define new Assign the size standard peaks as shown in Figure 13 in Section IX E Store the size standard in the Size Standards folder 7 Apply the size standard file to the samples then analyze the sample files See Section VI F for additional information on the use of the PowerTyper 16 Macro Release 2 0 and Genotyper software For additional information regarding the GeneScan analysis software refer to the GeneScan Analysis Software User s Manual 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 TMD012 Revised 5 08 Analysis Range Start Defined in Step 2 Stop 10 000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light1 Peak Detection Peak Amplitude Thresholds2 B Y G R Min Peak Half Width 2pts Size Call Range Min 60 Max 600 Size Calling Method Local Southern Method Split Peak Correction None 1Smooth options should be determined by individual laboratories Occasionally the TH01 alleles 9 3 and 10 will not be distinguished using heavy smoothing 2The peak amplitude thresholds are the minimum peak heights that the software will call as a peak Value
3. 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 9 Page 10 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 TMD012 Printed in USA Revised 5 08 Protocol for the GeneAmp PCR System 9700 Thermal Cycler1 Protocol for the GeneAmp PCR System 2400 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 100 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 100 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak Protocol for the GeneAmp PCR System 9600 Thermal Cycler Protocol for the Perkin Elmer Model 480 Thermal Cycler 95 C for 11 minutes then 96 C for 1 minute th
4. and is licensed to Whatman GenBank is a registered trademark of the U S Dept of Health and Human Services 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 Cambrex Corporation Macintosh is a registered trademark of Apple Computer Inc Microsoft Windows and Windows NT are registered trademarks of Microsoft Corporation Nalgene is a registered trademark of Nalge Nunc International Triton is a registered trademark of Union Carbide Chemicals and Plastics Technology 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 tmd012 0508 qxp 9 16 2008 3 48 PM Page 58
5. 6 Denature samples and ladder by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading 7 Assemble the tubes in the appropriate autosampler tray 48 or 96 tube 8 Place the autosampler tray in the instrument and close the instrument doors Instrument Preparation Refer to the instrument users manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 1 Open the ABI PRISM 310 data collection software 2 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 For rows containing PowerPlex 16 Allelic Ladder Mix insert the word ladder in the sample info column for the blue dye color yellow dye color and green dye color This information must be entered to successfully analyze your data using the PowerTyper 16 Macro Release 2 0 3 Create a new GeneScan injection list Select the appropriate sample sheet by using the pull down menu 4 Select the GS STR POP4 1ml A Module using the pull down menu Change the injection time to 3 seconds and the run time to 30 minutes 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 30 You m
6. Allelic ladder and primer pair mix were not compatible Ensure the same as samples that the allelic ladder is from the same kit as the primer pair mix Buffer incompatibility Samples were diluted in the wrong buffer Use Gold ST R 1X Buffer to dilute samples Poor quality formamide Use only Hi Di formamide when analyzing samples Be sure the allelic ladder and samples are from the same instrument run Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to determine sizes Poor injection of allelic ladder Include more than one ladder per instrument run Peak height imbalance Excessive amount of DNA Amplification of gt 1ng of template can result in an imbalance with smaller loci showing more product than larger loci Use less template or reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling to improve locus to locus balance Note Dilution of overamplified samples can result in dropout of larger loci Use of FTA paper Results may be similar to those obtained with excess amounts of DNA template Reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling to improve locus to locus balance Degraded DNA sample DNA template is degraded and larger loci show diminished yield Repurify the template DNA Insufficie
7. Allelic ladder peaks are GeneMapper ID software was not used or microsatellite labeled off ladder analysis settings were used instead of HID analysis settings GeneMapper software does not use the same algorithms as GeneMapper ID software and cannot correct for sizing differences using the allelic ladder Promega recommends using GeneMapper ID software to analyze PowerPlex reactions If using GeneMapper ID software version 3 2 be sure that the analysis method selected is an HID method This can be verified by opening the analysis method using the GeneMapper Manager then selecting the General tab The analysis type cannot be changed If the method is not HID it should be deleted and a new analysis method created tmd012 0508 qxp 9 16 2008 3 48 PM Page 42 Page 43 VII C PowerTyper 16 Macro Symptoms Causes and Comments File does not open Genotyper software was not installed Be certain that the on your computer Genotyper software version 2 5 Macintosh or version 3 6 or higher Windows NT is installed Incorrect version of Genotyper software The PowerTyper 16 Macro will not work with Genotyper software versions prior to version 2 5 The CD ROM may have been damaged during shipment Contact Technical Services by e mail genetic promega com The file was corrupted during download or transfer Download the file again or obtain the file on CD ROM Error message Allelic ladder sample files were not
8. Biosystems 1 5ml amber colored microcentrifuge tubes Fisher Cat 05 402 26 aerosol resistant pipette tips see Section IX I AmpliTaq Gold DNA polymerase Applied Biosystems Nuclease Free Water Cat P1193 Mineral Oil Cat DY1151 for use with the model 480 thermal cycler We routinely amplify 0 5 1ng of template DNA in a 25 l reaction volume using the protocols detailed below Preferential amplification of smaller loci can occur Expect to see high peak heights at the smaller loci and relatively lower peak heights at the larger loci if more than the recommended amount of template is used Reduce the amount of template DNA or the number of cycles to correct this The PowerPlex 16 System is optimized for the GeneAmp PCR System 9700 thermal cycler Amplification protocols for the GeneAmp PCR Systems 9600 and 2400 thermal cyclers and Perkin Elmer model 480 thermal cycler are provided IV A Amplification Setup The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and postamplification reagents in separate rooms Prepare amplification reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for amplification setup Meticulous care must be taken to ensure successful amplification A guide to amplification troubleshooting is provided in Section VII A 1 Thaw the Gold ST R 10X Buffer and Power
9. DC6730 PowerPlex Y System 50 reactions DC6761 200 reactions DC6760 Not for Medical Diagnostic Use Accessory Components Product Size Cat PowerPlex Matrix Standards 310 50 l each dye DG4640 PowerPlex Matrix Standards 3100 3130 25 l each dye DG4650 PowerTyper Macros 1 CD ROM DG3470 Internal Lane Standard 600 150 l DG1071 Gold ST R 10X Buffer 1 2ml DM2411 Mineral Oil 12ml DY1151 Nuclease Free Water 50ml 2 25ml P1193 Not for Medical Diagnostic Use For Laboratory Use Sample Preparation Systems Product Size Cat DNA IQ System 100 reactions DC6701 400 reactions DC6700 Differex System 50 samples DC6801 200 samples DC6800 Maxwell 16 Instrument each AS2000 DNA IQ Reference Sample Kit for Maxwell 16 48 preps AS1040 DNA IQ Casework Sample Kit for Maxwell 16 48 preps AS1210 Plexor HY System 800 reactions DC1000 200 reactions DC1001 Slicprep 96 Device 10 pack V1391 Not for Medical Diagnostic Use For Laboratory Use For Research Use Only Not for use in diagnostic procedures tmd012 0508 qxp 9 16 2008 3 48 PM Page 56 Page 57 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 TMD012 Revised 5 08 Polyacrylamide Gel Electrophoresis Reag
10. Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers PowerPlex Matrix Standards 3100 3130 See Section IX I for ordering information III Before You Begin III A Precautions The application of PCR based typing for forensic or paternity casework requires validation studies and quality control measures that are not contained in this manual 11 12 The quality of the purified DNA as well as small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect PCR success We suggest strict adherence to recommended procedures for amplification and fluorescence detection PCR based STR analysis is subject to contamination by very small amounts of nontemplate human DNA Extreme care should be taken to avoid cross contamination when preparing sample DNA handling primer pairs assembling amplification reactions and analyzing amplification products Reagents and materials used prior to amplification Gold ST R 10X Buffer and PowerPlex 16 10X Primer Pair Mix are provided in a separate box and should be stored separately from those used following amplification PowerPlex 16 Allelic Ladder Mix and Internal Lane Standard 600 Always include a negative control reaction i e no template to detect reagent contamination We highly recommend the use of gloves and aerosol resistant pipette tips e g ART tips Section IX I Some reagents used in the
11. In addition low level artifacts in the TMR channel may be observed between 142 144 and 400 405bp These artifacts are not template derived and may appear in the negative control and in low product yield analyses The peak heights of these artifacts may increase with longer injection time or higher injection voltage VII Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com VII A Amplification and Fragment Detection Symptoms Causes and Comments Faint or absent allele peaks Impure template DNA Because of the small amount of template used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors might be present in the DNA sample Insufficient template Use the recommended amount of template DNA Insufficient template Low copy number LCN analysis using capillary electrophoresis may benefit from reducing competing charged particles during injection This can be accomplished with postPCR cleanup or desalting lower conductivity formamide or reduced amounts of ILS 600 In house validation should be performed for any of these methods Insufficient enzyme activity Use the recommended amount of AmpliTaq Gold DNA polymerase Check the expiration date on the tube label Incorrect amplification program Confirm the amplification program High salt concen
12. Positive Control or Negative Control Every folder in the project must contain at least one ladder that is designated as such for proper genotyping 3 In the Analysis Method column select the analysis method created in the Creating a Databasing or Paternity Analysis Method section 4 In the Panel column select PowerPlex_16_ID3 2 X where X refers to the most recent version of the panel files This is the panel set that was imported in Section VI A 5 In the Size Standard column select the size standard that was created in the Creating a Size Standard section 6 If analyzing data from an ABI PRISM 310 Genetic Analyzer or an ABI PRISM 377 DNA Sequencer ensure that the appropriate matrix file is selected in the Matrix column 7 Select Analyze green arrow button to start the data analysis VI D Sample Analysis Using the GeneScan Software and PC Operating Systems 1 Analyze data using the GeneScan analysis software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start position in the analysis parameters 3 The recommended analysis parameters are shown in Figure 8 4 The analysis pa
13. Printed in USA Part TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 13 Page 14 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 TMD012 Printed in USA Revised 5 08 V B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 continued Caution Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 injections 9 5 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of form
14. Products 56 I Description STR short tandem repeat loci consist of short repetitive sequence elements 3 7 base pairs in length 1 4 These repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers which may be detected using the polymerase chain reaction 5 8 Alleles of STR loci are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using radioactive silver stain or fluorescence detection following electrophoretic separation The PowerPlex 16 System a d 9 10 allows co amplification and three color detection of sixteen loci fifteen STR loci and Amelogenin including Penta E D18S51 D21S11 TH01 D3S1358 FGA TPOX D8S1179 vWA Amelogenin Penta D CSF1PO D16S539 D7S820 D13S317 and D5S818 One primer for each of the Penta E D18S51 D21S11 TH01 and D3S1358 loci is labeled with fluorescein FL one primer for each of the FGA TPOX D8S1179 vWA and Amelogenin loci is labeled with carboxy tetramethylrhodamine TMR and one primer for each of the Penta D CSF1PO D16S539 D7S820 D13S317 and D5S818 loci is labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxy fluorescein JOE All sixteen loci are amplified simultaneously in a single tube and analyzed in a single injection or
15. Promega Corporation 245 69 tmd012 0508 qxp 9 16 2008 3 48 PM Page 44 Page 45 12 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 13 Budowle B et al 1991 Analysis of the VNTR locus D1S80 by the PCR followed by high resolution PAGE Am J Hum Genet 48 137 44 14 Nakamura Y et al 1987 Variable number of tandem repeat VNTR markers for human gene mapping Science 235 1616 22 15 Budowle B and Monson K L 1989 In Proceedings of an International Symposium on the Forensic Aspects of DNA Analysis Government Printing Office Washington DC 16 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 17 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 18 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 19 Magnuson V L et al 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase Implications for PCR based genotyping BioTechniques 21 700 9 20 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 12 21 M
16. column 7 Select Analyze green arrow button to start data analysis VI C Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software 1 Select Tools then GeneMapper Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Promega Technical Services by e mail genetic promega com for assistance 5 Enter a descriptive name for the analysis method such as PowerPlex16_20 filter 6 Select the Allele tab 7 Select the bin set corresponding to the PowerPlex System Promega_Bins_ID3 2 X where X refers to the most recent version of the bin set 8 Ensure that the Use marker specific stutter ratio if available box is checked 9 Enter the values shown in Figure 7 for proper filtering of peaks when using the PowerPlex 16 System For an explanation of the proper usage and effect of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2
17. gel lane The PowerPlex 16 Monoplex System Penta E Fluorescein Cat DC6591 and PowerPlex 16 Monoplex System Penta D JOE Cat DC6651 are available to amplify the Penta E and Penta D loci respectively Each monoplex system allows amplification of a single locus to confirm results obtained with the PowerPlex 16 System PowerPlex 16 BIO System or PowerPlex 2 1 System The monoplex systems can be also used to re amplify DNA samples when one or more of the loci do not amplify initially due to nonoptimal amplification conditions or poor DNA template quality The PowerPlex 16 System is compatible with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers Applied Biosystems 3130 and 3130xl Genetic Analyzers and ABI PRISM 377 DNA Sequencer The protocols presented in this manual were tested at Promega Corporation Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection time or loading volume for each laboratory instrument In house validation should be performed tmd012 0508 qxp 9 16 2008 3 47 PM Page 2 Page 3 The PowerPlex 16 System provides all of the materials necessary for amplification of STR regions of purified genomic DNA except for AmpliTaq Gold DNA polymerase This manual contains separate protocols for use of the PowerPlex 16 System with the Perkin Elmer model 480 and GeneAmp PCR system 9600 9700 and 2400 thermal cyclers in additi
18. in the 270 271bp position in the JOE dye channel One or more extra peaks that are not directly related to amplification may be observed at positions 8 26 bases smaller than TPOX alleles and 6 21 bases smaller than vWA alleles These extra peaks occur when the amplified peaks are particularly intense high signal level or template amount formamide polymer or capillary was of poor quality or denaturation was ineffective Please see Section VII for more information on how to minimize these artifacts Page 36 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 TMD012 Printed in USA Revised 5 08 5682TA A B C Figure 10 The PowerPlex 16 Allelic Ladder Mix The PowerPlex 16 Allelic Ladder Mix was analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection The sample file was analyzed with the GeneMapper ID software version 3 2 and PowerPlex 16 panel and bin files Panel A The fluorescein labeled allelic ladder components and their allele designations Panel B The JOE labeled allelic ladder components and their allele designations Panel C The TMR labeled allelic ladder components and their allele designations tmd012 0508 qxp 9 16 2008 3 48 PM Page 36 Page 37 A low level artifact in the D5S818 region of the JOE channel may be observed between 114 120bp
19. pair size of alleles in the allelic ladder are outside of the defined category range Be sure internal lane standard fragments are correctly sized Redefine internal lane standard fragments and re analyze the sample using GeneScan software Compare the size of the smallest allele in the allelic ladder with the base pair size and range listed in the categories for the same alleles If necessary increase the category start range in the category window to greater than 6bp and save the macro under a new name Allelic ladder peaks were too high causing stutter peaks to be called as allele peaks Use a shorter injection time decrease the amount of allelic ladder used or re analyze the allelic ladder sample using increased peak amplitude thresholds in the GeneScan analysis parameters 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 48 PM Page 43 Page 44 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 TMD012 Printed in USA Revised 5 08 VII C PowerTyper 16 Macro continued Symptoms Causes and Comments The plots window or allele The macros were not run in the proper order Use the POWER table does not di
20. reaction tubes 4 Add the final volume of each reagent listed in Table 2 into a sterile 1 5ml amber colored tube Mix gently Table 2 shows the component volumes per reaction A worksheet to calculate the required amount of each PCR master mix component is provided in Section IX F Table 10 Amplification of gt 1ng of DNA template results in an imbalance in peak heights from locus to locus The smaller loci show greater amplification yield than the larger loci Reducing the number of cycles in the amplification program by 2 to 4 cycles i e 10 20 or 10 18 cycling can improve locus to locus balance 5 Pipet PCR master mix into each reaction tube 6 Pipet the template DNA 0 5 1ng for each sample into the respective tube containing PCR master mix Page 8 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 TMD012 Printed in USA Revised 5 08 Table 2 PCR Master Mix for the PowerPlex 16 System PCR Master Mix Component1 Volume Per Reaction nuclease free water to a final volume of 25 0 l Gold ST R 10X Buffer 2 5 l PowerPlex 16 10X Primer Pair Mix 2 5 l AmpliTaq Gold DNA polymerase2 0 8 l 4u template DNA 0 5 1ng 3 up to 19 2 l total reaction volume 25 l 1Add nuclease free water to the PCR master mix first then add Gold ST R 10X Buffer PowerPlex 16 10X
21. the GeneScan Analysis Software User s Manual Enter the appropriate sample information in the sample info column For lanes containing PowerPlex 16 Allelic Ladder Mix insert the word ladder in the sample info column for the blue dye color yellow dye color and green dye color This information must be entered to successfully analyze your data using the PowerTyper 16 Macro Release 2 0 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 by using the pull down menus 5 Select the appropriate gel matrix file Section III B Gel Pre Run 1 Remove 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 the plate check 5 Ad
22. the PowerPlex 1 2 and 16 Systems in Various Populations Matching Probability STR System African American Caucasian American Hispanic American Asian American PowerPlex 1 2 System 8 STR loci 1 in 2 77 108 1 in 1 15 108 1 in 1 45 108 1 in 1 32 108 PowerPlex 16 System 15 STR loci 1 in 1 41 1018 1 in 1 83 1017 1 in 2 93 1017 1 in 3 74 1017 Table 8 Typical Paternity Indices of the PowerPlex 1 2 and 16 Systems in Various Populations Typical Paternity Index STR System African American Caucasian American Hispanic American Asian American PowerPlex 1 2 System 497 262 318 471 PowerPlex 16 System 2 510 000 1 520 000 522 000 4 110 000 Table 9 Power of Exclusion of the PowerPlex 1 2 and 16 Systems in Various Populations Power of Exclusion STR System African American Caucasian American Hispanic American Asian American PowerPlex 1 2 System 0 9982042 0 9968863 0 9973367 0 9981793 PowerPlex 16 System 0 9999996 0 9999994 0 9999983 0 9999998 tmd012 0508 qxp 9 16 2008 3 48 PM Page 51 Page 52 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 TMD012 Printed in USA Revised 5 08 IX D DNA Extraction and Quantitation Methods The DNA IQ System Cat DC6700 is a DNA isolation and quantitation system designed specifically fo
23. the ethidium bromide solution and replace with deionized water Allow the gel to destain for 20 minutes 9 Photograph the gel using a UV transilluminator 302nm Note When analyzing the data do not be alarmed by extra bands in addition to alleles DNA heteroduplexes can be expected when performing nondenaturing agarose gel electrophoresis The sole purpose of the agarose gel is to confirm PCR success tmd012 0508 qxp 9 16 2008 3 48 PM Page 54 Page 55 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 TMD012 Revised 5 08 IX H Composition of Buffers and Solutions 10 ammonium persulfate Add 0 05g of ammonium persulfate to 500 l of deionized water Blue Dextran Loading Solution 88 25 formamide 15mg ml blue dextran 4 1mM EDTA pH 8 0 ethidium bromide solution 10mg ml 1 0g ethidium bromide Dissolve ethidium bromide in 100ml of deionized water Wrap in aluminum foil or transfer the solution to a dark bottle and store at room temperature Caution Ethidium bromide is a powerful mutagen Wear gloves when working with the dye and wear a mask when weighing it Gold ST R 10X Buffer 500mM KCl 100mM Tris HCl pH 8 3 at 25 C 15mM MgCl2 1 Triton X 100 2mM each dNTP 1 6mg ml BSA TAE 50X buffer pH 7 2 242g Tris base 57 1ml glacial acetic a
24. 008 3 47 PM Page 27 Page 28 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 TMD012 Printed in USA Revised 5 08 VI C Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software continued Creating a Size Standard 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 5 The type of analysis method selected must match the type of analysis method created earlier Select OK 5 Enter a detailed name such as ILS 600 advanced in the Size Standard Editor Figure 6 6 Choose red as the color for the size standard dye 7 Enter the sizes of the internal lane standard fragments see Section IX E Figure 13 8 Select OK 5785TA Figure 7 The Allele tab with settings for using a 20 peak filter Select the bin set Promega_Bins_ID3 2 X txt where X refers to the most recent version of the bin set tmd012 0508 qxp 9 16 2008 3 47 PM Page 28 Page 29 Processing Data for Databasing or Paternity Samples 1 Import sample files into a new project as described in the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial 2 In the Sample Type column use the drop down menu to select Ladder Sample
25. 100 90 0 C 100 60 0 C 29 60 0 C 29 70 0 C 23 70 0 C 23 3 tmp 10 cycles 3 tmp 22 cycles tmd012 0508 qxp 9 16 2008 3 47 PM Page 10 Page 11 V Instrument Setup and Sample Preparation V A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath aerosol resistant pipette tips 3100 or 3130 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 or 3130 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 3100 3130 Cat DG4650 The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm 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 Caution Formamide is an irritant and a teratogen avoid in
26. 11 D13S317 8 8 11 11 11 11 D5S818 11 12 11 11 11 13 1Information on strains 9947A 9948 and K562 is available online at locus umdnj edu nigms Strain K562 is available from the American Type Culture Collection www atcc org Manassas VA Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 26 2Strain K562 displays three alleles at the D21S11 locus 3Strain 9948 displays three alleles at the CSF1PO locus The peak height for allele 12 is much lower than those for alleles 10 and 11 tmd012 0508 qxp 9 16 2008 3 48 PM Page 50 Page 51 A measure of discrimination often used in paternity analyses is the paternity index PI a means for presenting the genetic odds in favor of paternity given the genotypes for the mother child and alleged father 35 The typical paternity indices for the PowerPlex 1 2 and 16 Systems are shown in Table 8 The PowerPlex 16 System provides typical paternity indices exceeding 500 000 in each population group An alternative calculation used in paternity analyses is the power of exclusion 35 This value calculated for the PowerPlex 16 System exceeds 0 999998 in all populations tested Table 9 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 TMD012 Revised 5 08 Table 7 Matching Probabilities of
27. 17 CSF1PO JOE 321 357 6 15 D16S539 JOE 264 304 5 8 15 D7S820 JOE 215 247 6 14 D13S317 JOE 176 208 7 15 D5S818 JOE 119 155 7 16 1The length of each allele in the allelic ladder has been confirmed by sequence analyses 2When using an internal lane standard such as the Internal Lane Standard 600 the calculated sizes of allelic ladder components may differ from those listed This occurs because different sequences in allelic ladder and ILS components may cause differences in migration The dye label also affects migration of alleles 3The alleles listed are those with a frequency of gt 1 1000 4For a current list of microvariants see the Variant Allele Report published at the U S National Institute of Standards and Technology NIST web site at www cstl nist gov div831 strbase 5Amelogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band tmd012 0508 qxp 9 16 2008 3 48 PM Page 49 Page 50 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 TMD012 Printed in USA Revised 5 08 IX C Power of Discrimination The fifteen STR loci amplified with the PowerPlex 16 System provide powerful discrimination Population statistics for these loci and their various multiplex combinations are displayed in Tables 7 9 These data were gener
28. 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with the following exceptions 1 In the Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Confirm that the injection time is 5 seconds and the injection voltage is 3kV Lengthen the run time to 2 000 seconds Give a descriptive name to your run module and select OK Note Instrument sensitivities can vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 2 In the Protocol Manager select New Type a name for your protocol Select Regular in the Type drop down list and select the run module you created in the previous step in the Run Module drop down list Lastly select F in the Dye Set drop down list Select OK 3 In the Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select GeneMapper Generic in the Application drop down list and select the appropriate plate type 96 well Add entries in the owner and operator windows and select OK Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions Page 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA T
29. IS core loci in a single reaction The PowerPlex 16 System also contains two low stutter highly polymorphic pentanucleotide repeat loci Penta E and Penta D These additional loci add significantly to the discrimination power of the system making the PowerPlex 16 System a single amplification system with a power of exclusion sufficient to resolve paternity disputes definitively In addition the extremely low level of stutter seen with Penta E and Penta D makes them ideal loci to evaluate DNA mixtures often encountered in forensic casework Finally the Amelogenin locus is included in the PowerPlex 16 System to allow gender identification of each sample Table 6 lists the PowerPlex 16 System alleles revealed in commonly available standard DNA templates We have carefully selected STR loci and primers to avoid or minimize artifacts including those associated with Taq DNA polymerase such as repeat slippage and terminal nucleotide addition Repeat slippage 16 17 sometimes called n 4 bands stutter or shadow bands is due to the loss of a repeat unit during DNA amplification somatic variation within the DNA or both The amount of this artifact observed depends primarily on the locus and the DNA sequence being amplified Terminal nucleotide addition 18 19 occurs when Taq DNA polymerase adds a nucleotide generally adenine to the 3 ends of amplified DNA fragments in a template independent manner The efficiency w
30. Plex 16 10X Primer Pair Mix Notes 1 Mix reagents by vortexing for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix as this may cause the primers to be concentrated at the bottom of the tube 2 A precipitate may form in the Gold ST R 10X Buffer If this occurs warm the solution briefly at 37 C then vortex until the precipitate is in solution 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does waste a small amount of each reagent it ensures that you will have enough PCR master mix for all samples It also ensures that each reaction contains the same master mix 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 7 IV A Amplification Setup continued 3 Place one clean 0 2ml or 0 5ml reaction tube for each reaction into a rack and label appropriately Alternatively use a MicroAmp plate and label appropriately Note If using the GeneAmp PCR System 9600 9700 or 2400 thermal cyclers use 0 2ml MicroAmp 8 strip reaction tubes or MicroAmp plate For the Perkin Elmer model 480 thermal cycler we recommend 0 5ml GeneAmp thin walled
31. Primer Pair Mix and AmpliTaq Gold DNA polymerase The template DNA will be added at Step 6 2Assumes the AmpliTaq Gold DNA polymerase is at 5u l If the enzyme concentration is different the volume of enzyme must be adjusted accordingly 3Store DNA templates in nuclease free water or TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA added should not exceed 20 of the final reaction volume PCR amplification efficiency and quality can be greatly altered by changes in pH due to added Tris HCl available magnesium concentration due to chelation by EDTA or other PCR inhibitors which may be present at low concentrations depending on the source of the template DNA and the extraction procedure used tmd012 0508 qxp 9 16 2008 3 47 PM Page 8 Page 9 7 For the positive amplification control dilute 9947A DNA to 0 5ng in the desired template DNA volume Pipet 0 5ng of the diluted DNA into a reaction tube containing PCR master mix 8 For the negative amplification control pipet nuclease free water instead of template DNA into a reaction tube containing PCR master mix 9 If using the GeneAmp PCR System 9600 9700 or 2400 thermal cycler and MicroAmp reaction tubes or plates no addition of mineral oil to the reaction tubes is required However if using the model 480 thermal cycler and GeneAmp reaction tu
32. R Locus Label Chromosomal Location GenBank Locus and Locus Definition Repeat Sequence1 5 3 Penta E FL 15q NA AAAGA D18S51 FL 18q21 3 HUMUT574 AGAA 23 D21S11 FL 21q11 21q21 HUMD21LOC TCTA Complex 23 TH01 FL 11p15 5 HUMTH01 human tyrosine hydroxylase gene AATG 23 D3S1358 FL 3p NA TCTA Complex FGA TMR 4q28 HUMFIBRA human fibrinogen alpha chain gene TTTC Complex 23 TPOX TMR 2p24 2pter HUMTPOX human thyroid peroxidase gene AATG D8S1179 TMR 8q NA TCTA Complex 23 vWA TMR 12p12 pter HUMVWFA31 human von Willebrand factor gene TCTA Complex 23 Amelogenin2 TMR Xp22 1 22 3 and Y HUMAMEL human Y chromosomal gene for Amelogenin like protein NA Penta D JOE 21q NA AAAGA CSF1PO JOE 5q33 3 34 HUMCSF1PO human c fms proto oncogene for CSF 1 receptor gene AGAT D16S539 JOE 16q24 qter NA GATA D7S820 JOE 7q11 21 22 NA GATA D13S317 JOE 13q22 q31 NA TATC D5S818 JOE 5q23 3 32 NA AGAT 1The August 1997 report 24 25 of the DNA Commission of the International Society for Forensic Haemogenetics ISFH states 1 for STR loci within coding genes the coding strand shall be used and the repeat sequence motif defined using the first possible 5 nucleotide of a repeat motif and 2 for STR loci not associated with a coding gene the first database entry or original literature description shall be used 2Am
33. T e c h n i c a l M a n u a l PowerPlex 16 System INSTRUCTIONS FOR USE OF PRODUCTS DC6530 AND DC6531 PRINTED IN USA Revised 5 08 Part TMD012 tmd012 0508 qxp 9 16 2008 3 47 PM Page 1 Page 1 I Description 2 II Product Components and Storage Conditions 4 III Before You Begin 5 A Precautions 5 B Matrix Standardization or Spectral Calibration 6 IV Protocols for DNA Amplification Using the PowerPlex 16 System 7 A Amplification Setup 7 B Amplification Thermal Cycling 9 V Instrument Setup and Sample Preparation 11 A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and
34. amber and close the instrument door 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 21 Page 22 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 TMD012 Printed in USA Revised 5 08 V D Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer continued Gel Electrophoresis and Detection 1 After loading select Cancel to stop the pre run 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 600 base ILS fragment will have migrated past the laser 4 Track and extract the gel lanes 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 the plates to air dry Do not scrape plates with abrasive materials during this process Note Soap and oil may build up on plates resulting in gel extrusion or hazy background Soak plates in 2N HCl for 15 minutes t
35. amide 2 Mix for 10 15 seconds using a vortex mixer 3 Pipet 10 l of formamide internal lane standard mix into each well 4 Add 1 l of amplified sample or 1 l of allelic ladder mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Editor in the Tools menu to modify injection time or voltage in the run module If peak heights are higher than desired samples can be diluted in Gold ST R 1X Buffer before mixing with loading cocktail The use of too much template DNA may result in uneven allele peak heights across loci For best results use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 5 Centrifuge plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the ABI PRISM 3100 Genetic Analyzer User s Manual for instructions on cleaning the blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe 1 Open the ABI PRISM 3100 data collection software 2 Change the GeneSc
36. an36_POP4DefaultModule module run time to 2 000 seconds tmd012 0508 qxp 9 16 2008 3 47 PM Page 14 Page 15 3 Change the injection voltage to 3kV 4 Change the injection time to 11 seconds Note Instrument sensitivities can vary Injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 5 Save the module with a new name e g GeneScan36_POP4PowerPlex16_3kV_11secs_2000 Use this as the initial run module for all runs 6 Open a new plate record Name the plate and select GeneScan Select the plate size 96 well Select Finish 7 Complete the plate record spreadsheet for the wells you have loaded Enter appropriate information into the sample name and color info columns For allelic ladder samples insert the word ladder into the color info column for the blue yellow and green dye colors This information must be entered to successfully analyze data with the PowerTyper 16 Macro Release 2 0 8 In the BioLIMS Project column select 3100_Project1 from the pull down menu 9 In the Dye Set column select Z from the pull down menu 10 When using the ABI PRISM 3100 data collection software version 1 0 1 or 1 1 select GeneScan36_POP4PowerPlex16_3kV_11secs_2000 from the pull down menu in the Run Module 1 column 11 To collect the data without autoanalyzing
37. anager icon in the upper left tile navigation pane 4 Select File then Import Panels 5 Navigate to the saved panel and bin files Select Promega_Panels_ID3 2 X txt where X refers to the most recent version of the panel and bin files Select Import 6 In the navigation pane highlight the Promega_Panels_ID3 2 X folder that you just imported 7 Select File then Import Bin Set 8 Navigate to the saved panel and bin files Select Promega_Bins_ID3 2 X txt then Import 9 At the bottom of the Panel Manager window select Apply then OK The panel manager window will close automatically VI B Creating a Casework Analysis Method with GeneMapper ID Software These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 11 1 Select Tools then GeneMapper Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 Select HID and select OK Note If you do not see the HID option you do not have the GeneMapper ID software Contact Applied Biosystems 5 Enter a descriptive name for the analysis method such as PowerPlex16 advanced 6 Select the Allele tab Figure 3 7 Select the bin set corresponding to the PowerPlex System Promega_Bins_ID3 2 X where X refers to the most recent version of the b
38. analysis of STR products are potentially hazardous and should be handled accordingly Table 1 describes the potential hazards associated with such reagents 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 5 Page 6 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 TMD012 Printed in USA Revised 5 08 III B Matrix Standardization or Spectral Calibration Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers A matrix must be generated for each individual instrument 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 For best results the PowerPlex Matrix Standards 3100 3130 Cat DG4650 should not be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 is required for spectral calibration on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 a
39. ated as part of a collaboration 27 with The Bode Technology Group Springfield VA North Carolina Bureau of Investigation Raleigh NC Palm Beach County Sheriff s Office West Palm Beach FL Virginia Division of Forensic Science Richmond VA and Charlotte Mecklenburg Police Department Laboratory NC Data generation included analysis of over 200 individuals from African American Caucasian American and Hispanic American populations Data for Asian Americans include analysis of more than 150 individuals For additional population data for STR loci see references 28 33 Table 7 shows the matching probability 34 for the PowerPlex 1 2 and 16 Systems in various populations The matching probability of the PowerPlex 16 System ranges from 1 in 1 83 1017 for Caucasian Americans to 1 in 1 41 1018 for African Americans Table 6 The PowerPlex 16 System Allele Determinations in Commonly Available Standard DNA Templates Standard DNA Templates1 STR Locus K5622 9947A 99483 Penta E 5 14 12 13 11 11 D18S51 15 16 15 19 15 18 D21S11 29 30 31 30 30 29 30 TH01 9 3 9 3 8 9 3 6 9 3 D3S1358 16 16 14 15 15 17 FGA 21 24 23 24 24 26 TPOX 8 9 8 8 8 9 D8S1179 12 12 13 13 12 13 vWA 16 16 17 18 17 17 Amelogenin X X X X X Y Penta D 9 13 12 12 8 12 CSF1PO 9 10 10 12 10 11 12 D16S539 11 12 11 12 11 11 D7S820 9 11 10 11 11
40. atic assignment of genotypes using the Genotyper software After samples are amplified detected using the ABI PRISM 310 or 3100 Genetic Analyzer using data collection software version 1 0 1 or 1 1 or ABI PRISM 377 DNA Sequencer and analyzed using the GeneScan analysis software sample files can be imported into the Genotyper program and analyzed using the PowerTyper 16 Macro Release 2 0 The PowerTyper 16 Macro Release 2 0 is available upon request from Promega The PowerTyper 16 Macro Release 2 0 is provided on the PowerTyper Macros CD ROM Cat DG3470 The PowerTyper Macros can also be downloaded from the Promega web site at www promega com geneticidtools The PowerTyper 16 Macro Release 2 0 is used in conjunction with Macintosh Genotyper software version 2 5 and Windows NT Genotyper software version 3 6 or later The Genotyper software must be installed on your computer before the PowerTyper 16 Macro Release 2 0 can be used Be certain the sample info Macintosh computers or color info Windows NT operating systems column for each lane containing allelic ladder mix contains the word ladder The macro uses the word ladder to identify the sample file s containing allelic ladder Sample info can be added or modified after importing into the PowerTyper Macro Highlight the sample then select show dye lanes window in the Views menu 1 Transfer the Pow
41. ation 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 TMD012 Printed in USA Revised 5 08 V C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler 310 capillaries 47cm 50 m performance optimized polymer 4 POP 4 10X genetic analyzer buffer with EDTA sample tubes and septa aerosol resistant pipette tips Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 310 Cat DG4640 crushed ice or ice water bath The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm 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 Caution Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Prepare a loa
42. ay need to optimize the injection time for individual instruments Injection times of 2 5 seconds are suggested for samples that contain 1ng of template DNA Note Migration of fragments may vary slightly over the course of a long ABI PRISM 310 Genetic Analyzer run This may be due to changes in temperature or changes in the column When analyzing many samples injections of allelic ladder at different times throughout the run can aid in accurately genotyping samples 5 Select the appropriate matrix file Section III B 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 17 Page 18 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 TMD012 Printed in USA Revised 5 08 V C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer continued 6 To analyze data automatically select the auto analyze checkbox and the appropriate analysis parameters and size standard Refer to the ABI PRISM 310 Genetic Analyzer User s Manual for specific information on these options 7 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis s
43. bes add one drop of mineral oil to each tube before closing Note Allow the mineral oil to flow down the side of the tube and form an overlay to limit sample loss or cross contamination due to splattering IV B Amplification Thermal Cycling This manual contains protocols for use of the PowerPlex 16 System with the Perkin Elmer model 480 and GeneAmp PCR system 9600 9700 and 2400 thermal cyclers For information on other thermal cyclers please contact Promega Technical Services by e mail genetic promega com Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection time or loading volume for each laboratory instrument Testing at Promega Corporation shows that 10 22 cycles work well for 0 5 1ng of purified DNA templates For higher amounts of input DNA i e FTA paper or to decrease sensitivity fewer cycles such as 10 16 10 18 or 10 20 should be evaluated In house validation should be performed 1 Place the tubes or MicroAmp plate in the thermal cycler 2 Select and run a recommended protocol The preferred protocols for use with the GeneAmp PCR System 9600 9700 and 2400 thermal cyclers and Perkin Elmer model 480 thermal cycler are provided below 3 After completion of the thermal cycling protocol store the samples at 20 C in a light protected box Note Storage of amplified samples at 4 C or higher may produce degradation products
44. cid 100ml 0 5M EDTA stock Add Tris base and EDTA stock to 500ml of deionized water Add glacial acetic acid Bring the volume to 1 liter with deionized water TBE 10X buffer 107 8g Tris base 7 44g EDTA Na2EDTA 2H2O 55 0g boric acid Dissolve Tris base and EDTA in 800ml of deionized water Slowly add the boric acid and monitor the pH until the desired pH of 8 3 is obtained Bring the final volume to 1 liter with deionized water TE 4 buffer 10mM Tris HCl 0 1mM EDTA pH 8 0 2 21g Tris base 0 037g EDTA Na2EDTA 2H2O Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Bring the final volume to 1 liter with deionized water tmd012 0508 qxp 9 16 2008 3 48 PM Page 55 Page 56 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 TMD012 Printed in USA Revised 5 08 IX I Related Products Fluorescent STR Multiplex Systems Product Size Cat PowerPlex 16 Monoplex System Penta E Fluorescein 100 reactions DC6591 PowerPlex 16 Monoplex System Penta D JOE 100 reactions DC6651 PowerPlex ES Monoplex System SE33 JOE 100 reactions DC6751 PowerPlex 1 2 System 100 reactions DC6101 PowerPlex 16 BIO System 100 reactions DC6541 400 reactions DC6540 PowerPlex ES System 100 reactions DC6731 400 reactions
45. d TBE 1X buffer to the top and bottom buffer chambers of the instrument 6 Using a 60cc syringe filled with buffer remove any air bubbles from the well area of the gel and place the lid on the upper buffer chamber Using a syringe fitted with a bent 18 gauge needle remove any air bubbles from the bottom of the gel Page 20 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 TMD012 Printed in USA Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 20 Page 21 7 Attach the heating plate connect the water tubing attach all electrodes close the instrument door and select PreRun Allow the gel to pre run 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 samples and allelic ladder samples during the gel pre run Sample Preparation and Loading 1 Prepare a loading cocktail by combining and mixing ILS 600 and Blue Dextran Loading Solution as follows 0 5 l ILS 600 lanes 1 5 l Blue Dextran Loading Solution lanes Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks 2 Vortex for 10 15 seconds 3 Combine 2 0 l of prepared loading cocktail and 1 0 l of amplified sample Note Inst
46. designations were assigned to the allelic ladders Figure 10 in Section VI H Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations If the POWER macro is run a second time the software will use the second ladder if the POWER macro is run a third time the software will use the third ladder etc until all ladders in the project are used If an allelic ladder fails to be analyzed or if many off ladder alleles are found in the samples samples should be re analyzed using another ladder from the project 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 48 PM Page 33 Page 34 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 TMD012 Printed in USA Revised 5 08 VI F Sample Analysis Using the Genotyper Software and PowerTyper 16 Macro continued 7 Double click on the Display Fluorescein Data macro to display the blue dye for all sample injections lanes Scroll down to observe and edit as needed 8 Double click on the Display TMR Data macro to display the yellow dye for all sample injections lanes Scroll down to observe and ed
47. ding cocktail by combining Internal Lane Standard 600 ILS 600 and Hi Di formamide as follows 1 0 l ILS 600 injections 24 0 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too high we recommend altering the loading cocktail to contain 0 5 l of ILS 600 and 24 5 l of Hi Di formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Combine 25 0 l of prepared loading cocktail and 1 0 l of amplified sample Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased If peak heights are higher than desired samples can be diluted in Gold ST R 1X Buffer before mixing with loading cocktail This may result in uneven allele peak heights across loci For best results use less template DNA in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles i e 10 18 or 10 20 cycling tmd012 0508 qxp 9 16 2008 3 47 PM Page 16 Page 17 4 Combine 25 0 l of prepared loading cocktail and 1 0 l of PowerPlex 16 Allelic Ladder Mix 5 Centrifuge tubes briefly to remove air bubbles from the wells if necessary
48. e Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 continued 4 Add 1 l of amplified sample or 1 l of allelic ladder mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module If peak heights are higher than desired samples can be diluted in Gold ST R 1X Buffer before mixing with loading cocktail This may result in uneven allele peak heights across loci For best results use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 5 Centrifuge plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument Instrument Preparation Refer to the instrument users manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer Analyze samples as described in the user s manual for the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with data collection software version
49. eactions Final Volume l Gold ST R 10X Buffer 2 5 l PowerPlex 16 10X Primer Pair Mix 2 5 l AmpliTaq Gold DNA polymerase1 0 8 l 4u nuclease free water2 l Per tube template DNA volume2 0 25 1ng up to 19 2 l total reaction volume 25 l 1Assumes the AmpliTaq Gold DNA polymerase is at 5u l If the enzyme concentration is different the volume of enzyme must be adjusted accordingly 2The master mix volume and template DNA volume should total 25 l Consider the volume of template DNA and add nuclease free water to the master mix to bring the final volume of the final reaction to 25 l Figure 13 Internal Lane Standard 600 An electropherogram showing Internal Lane Standard 600 fragments 5751TA 1 200 1 000 800 600 400 200 0 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 600 tmd012 0508 qxp 9 16 2008 3 48 PM Page 53 Page 54 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 TMD012 Printed in USA Revised 5 08 IX G Agarose Gel Electrophoresis of Amplification Products Optional This procedure is optional if PCR is routinely performed in your laboratory Agarose gel electrophoresis can be used to rapidly confirm am
50. ected using AMP FLP 13 or VNTR 14 analysis STR typing is also amenable to a variety of rapid DNA purification techniques that are compatible with PCR but do not provide enough DNA of appropriate quality for Southern blot based analyses Amplification products generated with Promega STR products are generally of discrete and separable lengths This allows construction of allelic ladders containing fragments of the same lengths as several or all known alleles for each locus Visual or software based comparison between the allelic ladder and amplified samples of the same locus allows rapid and precise assignment of alleles Results obtained using the PowerPlex 16 System can be recorded in a digitized format allowing direct comparison with stored databases Population analyses do not require the use of arbitrarily defined fixed bins for population data 15 tmd012 0508 qxp 9 16 2008 3 48 PM Page 46 Page 47 IX B Advantages of Using the Loci in the PowerPlex 16 System The loci included in the PowerPlex 16 System Tables 4 and 5 have been selected because they satisfy the needs of several major standardization bodies throughout the world For example the United States Federal Bureau of Investigation FBI has selected 13 STR core loci for typing prior to searching or including submitting samples in CODIS Combined DNA Index System the U S national database of convicted offender profiles The PowerPlex 16 System amplifies all COD
51. ed bag for shipping This component should be moved to the postamplification box after opening Storage Conditions Store all components at 20 C in a nonfrost free freezer The PowerPlex 16 10X Primer Pair Mix PowerPlex 16 Allelic Ladder Mix and Internal Lane Standard 600 are light sensitive and must be stored in the dark We strongly recommend that pre amplification and postamplification reagents be stored and used separately with different pipettes tube racks etc tmd012 0508 qxp 9 16 2008 3 47 PM Page 4 Page 5 Available Separately Product Size Cat Blue Dextran Loading Solution 3ml DV4351 PowerTyper Macros Release 2 0 1 CD ROM DG3470 For Laboratory Use Not For Medical Diagnostic Use The PowerTyper Macros Release 2 0 for use with Genotyper software are available from Promega This CD ROM contains the file PowerTyper 16 Macro Release 2 0 for use with the PowerPlex 16 System The macros can be also downloaded at www promega com geneticidtools The proper panel and bin files for use with GeneMapper ID software can be obtained from the Promega web site at www promega com geneticidtools panels_bins Matrix standards are required for initial setup of the color separation matrix The matrix standards are sold separately and are available for the ABI PRISM 310 Genetic Analyzer and 377 DNA Sequencer PowerPlex Matrix Standards 310 and the ABI PRISM 3100 and 3100 Avant
52. egory Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Category Allele 1 Category Allele 2 Sample Info Category Peak 1 Peak 2 tmd012 0508 qxp 9 16 2008 3 48 PM Page 34 Page 35 VI G Controls 1 Observe the results for the negative control The negative control should be devoid of amplification products 2 Observe the results for the 9947A positive control DNA Compare the 9947A DNA allelic repeat sizes with the locus specific allelic ladder The expected 9947A DNA allele designations for each locus are listed in Table 6 Section IX B VI H Results Representative results of the PowerPlex 16 System are shown in Figure 9 The PowerPlex 16 Allelic Ladder Mix is shown in Figure 10 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 TMD012 Revised 5 08 5683TA A B C D Figure 9 The PowerPlex 16 System A single template DNA 1 0ng was amplified using the PowerPlex 16 10X Primer Pair Mix Amplification products were mixed with Internal Lane Standard 600 and analyzed with an Applied Biosystems 3130 Genetic Analyzer using a 3kV 5 second injection Results were analyzed using GeneMapper ID software version 3 2 Panel A An electropherogram showing the peaks of the fluorescein labeled l
53. elogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band 9947A DNA female displays only the 106 base X specific band TMR carboxy tetramethylrhodamine FL fluorescein JOE 6 carboxy 4 5 dichloro 2 7 dimethoxyfluorescein NA not applicable tmd012 0508 qxp 9 16 2008 3 48 PM Page 48 Page 49 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 TMD012 Revised 5 08 Table 5 The PowerPlex 16 System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Components1 2 bases Repeat Numbers of Allelic Ladder Components Repeat Numbers of Alleles Not Present in Allelic Ladder3 4 Penta E FL 379 474 5 24 20 3 D18S51 FL 290 366 8 10 10 2 11 13 13 2 14 27 D21S11 FL 203 259 24 24 2 25 25 2 26 28 28 2 29 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 38 TH01 FL 156 195 4 9 9 3 10 11 13 3 D3S1358 FL 115 147 12 20 FGA TMR 322 444 16 18 18 2 19 19 2 20 20 2 21 21 2 22 22 2 23 23 2 24 24 2 25 25 2 26 30 31 2 43 2 44 2 45 2 46 2 TPOX TMR 262 290 6 13 D8S1179 TMR 203 247 7 18 vWA TMR 123 171 10 22 Amelogenin5 TMR 106 112 X Y Penta D JOE 376 449 2 2 3 2 5 7
54. en 94 C for 30 seconds ramp 68 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 10 cycles then 90 C for 30 seconds ramp 60 seconds to 60 C hold for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 22 cycles then 60 C for 30 minutes 4 C soak 95 C for 11 minutes then 96 C for 2 minutes then 94 C for 1 minute 60 C for 1 minute 70 C for 1 5 minutes for 10 cycles then 90 C for 1 minute 60 C for 1 minute 70 C for 1 5 minutes for 22 cycles then 60 C for 30 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the ramp rates indicated in the cycling program must be set and the program must be run in 9600 ramp mode The ramp rates are set in the Ramp Rate Modification screen While viewing the cycling program navigate to the Ramp Rate Modification screen by selecting More then Modify On the Ramp Rate Modification screen the default rates for each step are 100 The rate under each hold step is the rate at which the temperature will change to that hold temperature Figure 2 shows the ramp rates for the GeneAmp PCR System 9700 thermal cycler The ramp mode is set after start has been selected for the thermal cycling run A Select Method Options screen appears Select 9600 ramp mode and enter the reaction volume Figure 2 The ramp rates for the GeneAmp PCR System 9700 thermal cycler 7486MA 94 0 C
55. ents Product Size Cat Ammonium Persulfate 25g V3131 TBE Buffer 10X 1L V4251 Urea 1kg V3171 Blue Dextran Loading Solution 3ml DV4351 For Laboratory Use ART Aerosol Resistant Tips Product Volume Size tips pack Cat ART 10 Ultramicro Pipet Tip 0 5 10 l 960 DY1051 ART 20E Ultramicro Pipet Tip 0 5 10 l 960 DY1061 ART 20P Pipet Tip 20 l 960 DY1071 ART GEL Gel Loading Pipet Tip 100 l 960 DY1081 ART 100 Pipet Tip 100 l 960 DY1101 ART 100E Pipet Tip 100 l 960 DY1111 ART 200 Pipet Tip 200 l 960 DY1121 ART 1000E Pipet Tip 1 000 l 800 DY1131 tmd012 0508 qxp 9 16 2008 3 48 PM Page 57 Page 58 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 TMD012 Printed in USA Revised 5 08 a STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other patents issued to the Max Planck Gesellschaft zur F rderung der Wissenschaften e V Germany The development and use of STR loci are covered by U S Pat No 5 364 759 Australian Pat No 670231 and other pending patents assigned to Baylor College of Medicine Houston Texas Patents for the foundational PCR process European Pat Nos 201 184 and 200 362 expired on March 28 2006 In the U S the patents covering the foundational PCR process ex
56. erTyper 16 Macro Release 2 0 from the PowerTyper Macros CD ROM Cat DG3470 to a designated location on your computer hard drive Alternatively download the PowerTyper 16 Macro Release 2 0 from the Promega web site 2 Open the Genotyper software then the PowerTyper 16 Macro Release 2 0 For questions about the Genotyper software refer to the Genotyper Analysis Software User s Manual tmd012 0508 qxp 9 16 2008 3 48 PM Page 32 Page 33 3 In the File menu select Import and import the GeneScan project or sample files to be analyzed Import the blue yellow green and red dye colors Note To select the dye colors to be imported select Set Preferences in the Edit menu 4 Double click on the Check ILS macro The macros are listed at the bottom left corner of the active window A plots window will be displayed to show the internal lane standard i e ILS 600 in the red dye color Scroll down to view and confirm that the internal lane standard fragment sizes are correct If necessary re analyze samples using the GeneScan software and redefine internal lane standard fragments Note The software uses one ladder sample to determine allele sizes The macro uses the first ladder sample imported for allele designations 5 For casework double click on the POWER macro The POWER macro identifies alleles in the ladder sample and calculates offsets for all loci This process may take severa
57. eric tandem repeats Am J Hum Genet 49 746 56 3 Edwards A et al 1992 Genetic variation at five trimeric and tetrameric tandem repeat loci in four human population groups Genomics 12 241 53 4 Warne D et al 1991 Tetranucleotide repeat polymorphism at the human actin related pseudogene 2 actbp2 detected using the polymerase chain reaction Nucleic Acids Res 19 6980 5 Ausubel F M et al 1996 Unit 15 The polymerase chain reaction In Current Protocols in Molecular Biology Vol 2 John Wiley and Sons NY 6 Sambrook J Fritsch E F and Maniatis T 1989 Chapter 14 In vitro amplification of DNA by the polymerase chain reaction In Molecular Cloning A Laboratory Manual Second Edition Cold Spring Harbor Laboratory Press Cold Spring Harbor New York 7 PCR Technology Principles and Applications for DNA Amplification 1989 Erlich H A ed Stockton Press New York NY 8 PCR Protocols A Guide to Methods and Applications 1990 Innis M A et al eds Academic Press San Diego CA 9 Krenke B et al 2002 Validation of a 16 locus fluorescent multiplex system J Forensic Sci 47 773 85 10 Budowle B et al 2001 STR primer concordance study Forensic Sci Int 124 47 54 11 Presley L A et al 1992 The implementation of the polymerase chain reaction PCR HLA DQ alpha typing by the FBI laboratory In The Third International Symposium on Human Identification 1992
58. g the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail genetic promega com tmd012 0508 qxp 9 16 2008 3 47 PM Page 1 Page 2 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 TMD012 Printed in USA Revised 5 08 IX Appendix 46 A Advantages of STR Typing 46 B Advantages of Using the Loci in the PowerPlex 16 System 47 C Power of Discrimination 50 D DNA Extraction and Quantitation Methods 52 E The Internal Lane Standard 600 52 F Preparing the PowerPlex 16 System Master Mix 53 G Agarose Gel Electrophoresis of Amplification Products Optional 54 H Composition of Buffers and Solutions 55 I Related
59. halation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide Sample Preparation 1 Prepare a loading cocktail by combining and mixing Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 injections 9 5 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di formamide If peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Pipet 10 l of formamide internal lane standard mix into each well 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 11 V A Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Softwar
60. hat was used No size standard was selected In the size standards column be sure to select the appropriate size standard Size standard was not correctly defined or size peaks were missing Redefine size standard to include only peaks present in your sample Terminating analysis early or using short run times will cause larger ladder peaks to be missing This will cause your sizing quality to be flagged as red and no allele sizes will be called 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 TMD012 Revised 5 08 5686TA Figure 12 An example showing improper assignment of size standard fragments in the GeneMapper ID software tmd012 0508 qxp 9 16 2008 3 48 PM Page 41 Page 42 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 TMD012 Printed in USA Revised 5 08 VII B GeneMapper ID Analysis Software continued Symptoms Causes and Comments Error message The bin set assigned to the analysis method may have been Both the Bin Set used in the deleted In the GeneMapper Manager select the Analysis Analysis Method and the Panel Methods tab and open the analysis method of interest Select must belong to the same the Alleles tab and select an appro
61. hen rinse thoroughly to remove any buildup VI Data Analysis VI A PowerPlex Panel and Bin Sets with GeneMapper ID Version 3 2 To facilitate analysis of data generated with the PowerPlex 16 System we have created panel and bin files to allow automatic assignment of genotypes using GeneMapper ID software version 3 2 We recommend that users of GeneMapper ID software version 3 2 complete the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial to familiarize themselves with proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading to version 3 2 Getting Started 1 Obtain the proper panel and bin files for use with GeneMapper ID from the Promega web site at www promega com geneticidtools panels_bins 2 Enter your contact information and select GeneMapper ID version 3 2 Select Submit 3 Select the PowerPlex Panels amp Bin Sets link and save the zip file to your computer 4 Open the files using the Windows WinZip program and save the unzipped files to a known location on your computer tmd012 0508 qxp 9 16 2008 3 47 PM Page 22 Page 23 Importing Panel and Bin Files These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 4 1 Open the GeneMapper ID software version 3 2 2 Select Tools then Panel Manager 3 Highlight the Panel M
62. identified Be certain the Could not complete the sample info or color info column for each lane containing Run Macro command because PowerPlex 16 Allelic Ladder Mix contains the word ladder no dye lanes are selected The macro uses the word ladder to identify sample files containing allelic ladder All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors Error message Peak heights for one or more alleles in the allelic ladder Could not complete the sample file were below 150RFU The allelic ladder categories Run Macro command are defined as having a minimum peak height of 150RFU If because the labeled peak peak heights of ladder alleles are below 150RFU the software could not be found will not be able to locate the allele peak Re run the allelic ladder using more sample or longer injection time to assure peak heights above 150RFU CE spikes in the allelic ladder sample were identified as alleles by the macro Use a different injection of allelic ladder TH01 9 3 and 10 alleles were not separated when using heavy smoothing in the GeneScan analysis parameters Use light smoothing in the GeneScan analysis parameters Allelic ladder data were not compatible with the PowerTyper file used Confirm that the PowerTyper Macro file matches the allelic ladder being used The base
63. in set 8 Ensure that the Use marker specific stutter ratio if available box is checked 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 23 Page 24 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 TMD012 Printed in USA Revised 5 08 VI B Creating a Casework Analysis Method with GeneMapper ID Software continued 9 Enter the values shown in Figure 3 for proper filtering of stutter peaks when using the PowerPlex 16 System For an explanation of the proper usage and effects of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Note Some of these settings have been optimized and are different from the recommended settings in the user bulletin 10 Select the Peak Detector tab We recommend the settings shown in Figure 4 Note Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on the data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper siz
64. ing of the internal lane standard 5724TA Figure 3 The Allele tab Select the bin set Promega_Bins_ID3 2 X txt where X refers to the most recent version of the bin set tmd012 0508 qxp 9 16 2008 3 47 PM Page 24 Page 25 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID user s manual for more information 12 Select the Quality Flags tab You may also change these settings 13 Select OK to save your settings Creating a Size Standard 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 5 The type of analysis method selected must match the type of analysis method created earlier Select OK 5 Enter a detailed name such as ILS 600 advanced in the Size Standard Editor Figure 6 6 Choose red as the color for the size standard dye 7 Enter the sizes of the internal lane standard fragments see Section IX E Figure 13 8 Select OK 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 TMD012 Revised 5 08 5723TA Figure 4 The Peak Detector tab tmd012 0508 qxp 9 16 2008 3 47 PM Page 25 Page 26 Promega Corporation 2800 Woods Hollow R
65. inted in USA Revised 5 08 VII A Amplification and Fragment Detection continued Symptoms Causes and Comments Extra peaks visible in one Contamination with another template DNA or previously or all color channels amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not completely denatured Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to loading the gel or capillary Artifacts of STR amplification PCR amplification of STR systems sometimes generates artifacts that appear as faint peaks one repeat unit smaller than the allele Stutter band peak heights can be high if the samples are overloaded See Section VI H for additional information on stutter and artifacts Artifacts of STR amplification PCR amplification of STR systems can result in artifacts that appear as peaks one base smaller than the allele due to incomplete addition of the 3 A residue Be sure to perform the 30 minute extension step at 60 C after thermal cycling Section IV B High background Load less amplification product or decrease injection time See Section V CE related artifacts spikes Minor voltage changes or urea crystals passing by the laser can 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 sa
66. it as needed 9 Double click on the Display JOE Data macro to display the green dye for all sample injections lanes Scroll down to observe and edit as needed 10 Create the appropriate table by selecting the PowerTable Make Allele Table or Make CODIS Table macro The three available table formats are shown below The PowerTable option allows up to four alleles per sample file Additional information such as low peak signal or high peak signal is also included The Allele Table and CODIS Table options include only two alleles per locus If more than two alleles are present at a locus the smallest alleles identified are included The Allele Table format displays the categories loci in columns while the CODIS table format displays the categories in rows These tables can be customized to fit needs To save data in tables go to the Table drop down menu highlight Export to File and save the file with the desired name and location The saved file can be viewed and analyzed using Microsoft Excel PowerTable Format Allele Table Format CODIS Table Format 11 Save the analyzed data Go to the File menu and select Save as The PowerTyper Macro is a Genotyper file and can be overwritten if Save is used instead of Save as Sample Info Sample Comment Category Peak 1 Peak 2 Peak 3 Peak 4 Over flow Low Signal Satura tion Edited Label Edited Row Sample Info Cat
67. ith which this occurs varies with different primer sequences Thus an artifact band one base shorter than expected i e missing the terminal addition is sometimes seen We have modified primer sequences and added a final extension step of 60 C for 30 minutes 20 to the amplification protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used The presence of microvariant alleles alleles differing from one another by lengths other than the repeat length complicates interpretation and assignment of alleles There appears to be a correlation between a high degree of polymorphism a tendency for microvariants and increased mutation rate 21 22 Thus FGA and D21S11 display numerous relatively common microvariants For reasons yet unknown the highly polymorphic Penta E locus does not display frequent microvariants Table 5 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 48 PM Page 47 Page 48 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 TMD012 Printed in USA Revised 5 08 Table 4 The PowerPlex 16 System Locus Specific Information ST
68. l minutes When completed a plots window will open to display the allelic ladders i e Penta E D18S51 D21S11 TH01 and D3S1358 Alternatively for databasing or paternity double click on the POWER 20 Filter macro This macro has a higher level of filtering than the standard POWER macro to reduce the need for manual editing of peak labels The POWER 20 Filter should not be used if mixtures may exist In general allelic ladders contain fragments of the same lengths as many known alleles for the locus Allelic ladder sizes and repeat units are listed in Table 5 Section IX B Analysis using GeneScan analysis software and Genotyper software allows allele determination by comparing amplified sample fragments with allelic ladders and internal lane standards When using an internal lane standard the calculated lengths of allelic ladder components might differ from those listed in the table This is due to differences in migration resulting from sequence differences between the allelic ladder fragments and internal size standard and is not a matter of concern 6 Double click on the Allelic Ladders macro A plots window will open to display the blue fluorescein dye allelic ladders i e Penta E D18S51 D21S11 TH01 and D3S1358 green JOE dye allelic ladders i e Penta E CSF1PO D16S539 D7S820 D13S317 and D5S818 and yellow TMR dye allelic ladders i e FGA TPOX D8S1179 vWA and Amelogenin Confirm that the correct allele
69. mples to confirm CE related artifacts contaminants Contaminants in the water used with the instrument or to dilute the 10X genetic analyzer buffer may generate peaks in the blue and green dye colors Use autoclaved water change vials and wash buffer reservoir Excessive amount of DNA Amplification of gt 2ng template can result in a higher number of stutter bands Use less template DNA or reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix has been applied to the samples For the ABI PRISM 310 Genetic Analyzer and 377 DNA Sequencer generate a new matrix and apply it to the samples For the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers perform a new spectral calibration and re run the samples Instrument sensitivities can vary Optimize the injection or gel loading conditions See Section V Long term storage of amplified sample in formamide can result in degradation Repeat sample preparation using fresh formamide The CE polymer was beyond its expiration date or polymer was stored at room temperature for more than one week Maintain instrumentation on a daily or weekly basis as recommended by the manufacturer tmd012 0508 qxp 9 16 2008 3 48 PM Page 38 Symptoms Causes and Comments Allelic ladder not running
70. nce heterogeneity at the polymorphic STR locus HUMTH01 AATG n and reassignment of alleles in population analysis using a locus specific allelic ladder Am J Hum Genet 53 953 8 30 Hammond H et al 1994 Evaluation of 13 short tandem repeat loci for use in personal identification applications Am J Hum Genet 55 175 89 31 Bever R A and Creacy S 1995 Validation and utilization of commercially available STR multiplexes for parentage analysis In Proceedings from the Fifth International Symposium on Human Identification 1994 Promega Corporation 61 8 32 Sprecher C J et al 1996 General approach to analysis of polymorphic short tandem repeat loci BioTechniques 20 266 76 33 Lins A M et al 1996 Multiplex sets for the amplification of polymorphic short tandem repeat loci silver stain and fluorescent detection BioTechniques 20 882 9 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 48 PM Page 45 Page 46 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 TMD012 Printed in USA Revised 5 08 VIII References continued 34 Jones D A 1972 Blood samples Probability of di
71. nd 3130xl Genetic Analyzers The PowerPlex Matrix Standards 310 Cat DG4640 cannot be used to generate a matrix on these instruments For protocols and additional information on matrix standardization see the PowerPlex Matrix Standards 310 Technical Bulletin TBD021 which is supplied with Cat DG4640 For protocols and additional information on spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 which is supplied with Cat DG4650 These manuals are available upon request from Promega or online at www promega com tbs Table 1 Hazardous Reagents Reagents for ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers Hazard formamide irritant teratogen Reagents for ABI PRISM 377 DNA Sequencer Hazard acrylamide Long Ranger gel solution suspected carcinogen toxic ammonium persulfate oxidizer corrosive formamide contained in the Blue Dextran Loading Solution irritant teratogen TEMED corrosive flammable urea irritant tmd012 0508 qxp 9 16 2008 3 47 PM Page 6 Page 7 IV Protocols for DNA Amplification Using the PowerPlex 16 System Materials to Be Supplied by the User model 480 or GeneAmp PCR System 9600 9700 or 2400 thermal cycler Applied Biosystems microcentrifuge 0 5ml GeneAmp or 0 2ml MicroAmp reaction tubes or MicroAmp optical 96 well reaction plate Applied
72. nt template DNA Use the recommended amount of template DNA Stochastic effects can occur when amplifying low amounts of template Miscellaneous balance problems Thaw the 10X Primer Pair Mix and Gold ST R 10X Buffer completely and vortex for 15 seconds before using Do not centrifuge the 10X Primer Pair Mix after mixing Calibrate thermal cyclers and pipettes routinely Using a 59 C annealing temperature instead of 60 C has been shown to improve balance in some instances Impure template DNA Inhibitors that may be present in forensic samples can lead to allele dropout or imbalance 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 TMD012 Revised 5 08 Page 39 tmd012 0508 qxp 9 16 2008 3 48 PM Page 39 Page 40 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 TMD012 Printed in USA Revised 5 08 VII B GeneMapper ID Analysis Software Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper ID software the analysis parameters and size standard must both have Basic or Advanced as the analysis type If they are different an error is obtained Figure 11 An insufficient number of ILS 600 fragments was defined Be sure to define a
73. oad Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD012 Printed in USA Revised 5 08 VI B Creating a Casework Analysis Method with GeneMapper ID Software continued 5726TA Figure 6 The Size Standard Editor 5725TA Figure 5 The Select Dye and Analysis Method window tmd012 0508 qxp 9 16 2008 3 47 PM Page 26 Page 27 Processing Sample Data for Casework 1 Import sample files into a new project as described in the Applied Biosystems GeneMapper ID Software Human Identification Analysis Tutorial 2 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control Every folder in the project must contain at least one ladder that is designated as such for proper genotyping 3 In the Analysis Method column select the analysis method created previously in the Creating a Casework Analysis Method section 4 In the Panel column select PowerPlex_16_ID3 2 X where X refers to the most recent version of the panel files This is the panel set that was imported in Section VI A 5 In the Size Standard column select the size standard that was created in the Creating a Size Standard section 6 If analyzing data from an ABI PRISM 310 Genetic Analyzer or an ABI PRISM 377 DNA Sequencer ensure that the appropriate matrix file is selected in the Matrix
74. oci D3S1358 TH01 D21S11 D18S51 and Penta E Panel B An electropherogram showing the peaks of the JOE labeled loci D5S818 D13S317 D7S820 D16S539 CSF1PO and Penta D Panel C An electropherogram showing the peaks of the TMR labeled loci Amelogenin vWA D8S1179 TPOX and FGA Panel D An electropherogram showing the 60bp to 500bp fragments of the Internal Lane Standard 600 tmd012 0508 qxp 9 16 2008 3 48 PM Page 35 Artifacts and Stutter Stutter bands are a common amplification artifact associated with STR analysis Stutter products are often observed one repeat unit below the true allele peak and occasionally two repeat units smaller or one repeat unit larger than the true allele peak Frequently alleles with a greater number of repeat units will exhibit a higher percent stutter The pattern and intensity of stutter may differ slightly between primer sets for the same loci The level of stutter was determined and published as part of the PowerPlex 16 System validation 9 In addition to stutter peaks other artifact peaks can be observed at some of the PowerPlex 16 System loci Low level products can be seen in the n 2 and n 2 positions two bases below and above the true allele peak respectively with some loci such as D21S11 Samples may show low level artifacts in the noncalling regions between the D7S820 and D13S317 allele ranges and between the D3S1358 and TH01 allele ranges Occasionally an off ladder artifact can be seen
75. ocol ABI PRISM 377 DNA Sequencer Section V D tmd012 0508 qxp 9 16 2008 3 47 PM Page 3 Page 4 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 TMD012 Printed in USA Revised 5 08 II Product Components and Storage Conditions Product Size Cat PowerPlex 16 System 100 reactions DC6531 Not For Medical Diagnostic Use Cat DC6531 contains sufficient reagents for 100 reactions of 25 l each Includes Pre amplification Components Box Blue Label 1 300 l Gold ST R 10X Buffer 1 250 l PowerPlex 16 10X Primer Pair Mix 25 l 9947A DNA 10ng l Postamplification Components Box Beige Label 1 25 l PowerPlex 16 Allelic Ladder Mix 1 150 l Internal Lane Standard ILS 600 1 Protocol Product Size Cat PowerPlex 16 System 400 reactions DC6530 Not For Medical Diagnostic Use Cat DC6530 contains sufficient reagents for 400 reactions of 25 l each Includes Pre amplification Components Box Blue Label 4 300 l Gold ST R 10X Buffer 4 250 l PowerPlex 16 10X Primer Pair Mix 25 l 9947A DNA 10ng l Postamplification Components Box Beige Label 4 25 l PowerPlex 16 Allelic Ladder Mix 4 150 l Internal Lane Standard ILS 600 1 Protocol The PowerPlex 16 Allelic Ladder Mix is provided in a separate seal
76. oll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD012 Printed in USA Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 12 Page 13 4 In the GeneMapper plate record enter sample names in the appropriate cells Scroll to the right In the Results group 1 column select the desired results group In the Instrument Protocol 1 column select the protocol you created in Step 2 Be sure this information is present for each row that contains a sample name Select OK Note To create a new results group select New in the drop down menu in the results group column Select the General tab and enter a name Select the Analysis tab and select GeneMapper Generic in the Analysis type drop down list 5 Place samples in the instrument and close the instrument doors 6 In the spectral viewer confirm that dye set F is active and set the correct active calibration for dye set F 7 In the run scheduler locate the plate record that you just created in Steps 3 and 4 and click once on the name to highlight it 8 Once the plate record is highlighted click the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples 9 When the plate record is linked to the plate the plate graphic will change from yellow to green and the green Run Instrument arrow becomes enabled 10 Click on the green Run Instrument arrow on
77. oller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD21S11 Int J Leg Med 106 319 23 22 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 23 Griffiths R et al 1998 New reference allelic ladders to improve allelic designation in a multiplex STR system Int J Legal Med 111 267 72 24 B r W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Int J Legal Med 110 175 6 25 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 26 Fr geau C J et al 1995 Characterization of human lymphoid cell lines GM9947 and GM9948 as intra and interlaboratory reference standards for DNA typing Genomics 28 184 97 27 Levadokou E N et al 2001 Allele frequencies for fourteen STR loci of the PowerPlex 1 1 and 2 1 multiplex systems and Penta D locus in Caucasians African Americans Hispanics and other populations of the United States of America and Brazil J Forensic Sci 46 736 61 28 Lins A M et al 1998 Development and population study of an eight locus short tandem repeat STR multiplex system J Forensic Sci 43 1168 80 29 Puers C et al 1993 Identification of repeat seque
78. omated workstations contact your local Promega Branch Office or Distributor contact information available at www promega com worldwide or e mail genetic promega com IX E The Internal Lane Standard 600 The Internal Lane Standard ILS 600 contains 22 DNA fragments of 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases in length Figure 13 Each fragment is labeled with carboxy X rhodamine CXR and can be detected separately as a fourth color in the presence of PowerPlex 16 amplified material The ILS 600 is designed for use in each gel lane or CE injection to increase precision in analyses when using the PowerPlex 16 System Protocols for preparation and use of this internal lane standard are provided in Section V tmd012 0508 qxp 9 16 2008 3 48 PM Page 52 Page 53 IX F Preparing the PowerPlex 16 System Master Mix A worksheet to calculate the required amount of each PCR master mix component is provided in Table 10 Multiply the volume l per reaction by the total number of reactions to obtain the final master mix volume l 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 TMD012 Revised 5 08 Table 10 Master Mix for the PowerPlex 16 System PCR Master Mix Component Volume Per Reaction Number of R
79. on to protocols for separation of amplified products and detection of separated material Figure 1 Protocols for operation of the fluorescence detection instruments should be obtained from the instrument manufacturer Information on other Promega fluorescent STR systems and detection of amplified STR fragments using silver staining is available upon request from Promega or online 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 Printed in USA Part TMD012 Revised 5 08 Amplification Setup Thermal Cycling Instrument Setup and Sample Preparation Data Analysis Section IV B Section V Section VI Section IV A GeneAmp PCR System 9700 GeneAmp PCR System 9600 GeneAmp PCR System 2400 Model 480 Thermal Cycler Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Section V A GeneMapper ID Software Versions 3 1 and 3 2 GeneScan Software and PC Operating Systems GeneScan Software and Macintosh Operating Systems ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 Section V A ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Section V B ABI PRISM 310 Genetic Analyzer Section V C Figure 1 An overview of the PowerPlex 16 System prot
80. on to the bottom of the plates and to prevent formation of bubbles 7 Insert a 36 well sharkstooth comb or 34 well square tooth comb between the glass plates Sharkstooth combs with 64 or 96 wells also may be used 8 Secure the comb with 3 evenly spaced clamps 9 Keep the remaining acrylamide solution as a polymerization control 10 Allow polymerization to proceed for at least 2 hours Check the polymerization control to be sure that polymerization has occurred Note The gel may 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 Printed in USA Part TMD012 Revised 5 08 Table 3 Preparation of a 5 Long Ranger Polyacrylamide Gel 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 may be used tmd012 0508 qxp 9 16 2008 3 47 PM Page 19 V D Detection of Amplified 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
81. p 9 16 2008 3 47 PM Page 18 Page 19 Polyacrylamide Gel Preparation 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 may be obtained from the instrument manufacturer 1 Thoroughly clean glass plates with hot water and a 1 Liqui Nox solution Rinse extremely well using deionized water Allow the 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 the plates together using binder clamps 4 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 ingredients listed in Table 3 Stir the solution until the urea has dissolved 4 Filter the acrylamide solution through a 0 2 micron filter e g Nalgene tissue culture filter and degas for 5 minutes 5 Add 35 l of TEMED and 250 l of fresh 10 ammonium persulfate to 50ml of acrylamide solution and mix gently 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 movement of soluti
82. pired on March 29 2005 b U S Pat Nos 6 238 863 and 6 767 703 and Korean Pat No 691195 have been issued to Promega Corporation for materials and methods for identifying and analyzing intermediate tandem repeat DNA markers Other patents are pending c U S Pat Nos 5 843 660 6 479 235 6 221 598 and 7 008 771 Australian Pat No 724531 Canadian Pat No 2 118 048 Korean Pat No 290332 Singapore Pat No 57050 and Japanese Pat No 3602142 have been issued to Promega Corporation for multiplex amplification of STR loci Other patents are pending d The purchase of this product does not convey a license to use AmpliTaq Gold DNA polymerase You should purchase AmpliTaq Gold DNA polymerase licensed for the forensic and human identity field directly from your authorized enzyme supplier 2000 2008 Promega Corporation All Rights Reserved Maxwell Plexor and PowerPlex are registered trademarks of Promega Corporation Differex DNA IQ PowerTyper and Slicprep are trademarks of Promega Corporation ABI PRISM GeneMapper GeneScan Genotyper and MicroAmp are registered trademarks of Applera Corporation AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems Inc ART is a registered trademark of Molecular Bio Products Inc Biomek is a registered trademark of Beckman Coulter Inc Freedom EVO is a registered trademark of Tecan AG Corporation FTA is a registered trademark of Flinders Technologies Pty Ltd
83. plification success prior to performing polyacrylamide gel or capillary electrophoresis Materials to Be Supplied by the User Solution compositions are provided in Section IX H TAE 1X buffer agarose 5X loading solution ethidium bromide solution 0 5 g ml 1 Prepare a 2 agarose gel approximately 150cm2 by adding 2 0g of agarose to 100ml of TAE 1X buffer Mark the liquid level on the container then boil or heat in a microwave oven to dissolve the agarose Add preheated 60 C deionized water to make up for any volume lost due to evaporation 2 Cool agarose to 55 C before pouring into the gel tray Be sure that the gel tray is level Pour the agarose into the tray insert the gel comb and allow to set for 20 30 minutes 3 Prepare samples by mixing 10 l of each amplified sample with 2 5 l of 5X loading solution 4 Prepare 1 liter of TAE 1X buffer for the electrophoresis running buffer 5 Place the gel and tray in the electrophoresis gel box Pour enough running buffer into the tank to cover the gel to a depth of at least 0 65cm Gently remove the comb 6 Load each sample mixed with 5X loading solution prepared in Step 3 7 Set the voltage at 5 volts cm measured as the distance between the two electrodes Allow the gel to run for 2 hours 8 After electrophoresis stain the gel in TAE 1X buffer containing 0 5 g ml ethidium bromide Gently rock for 20 minutes at room temperature Remove
84. priate bin set Chemistry Kit The wrong bin set was chosen in the analysis method Allele tab Be sure to choose the appropriate bin set as shown in Figure 3 Significantly raised baseline Poor spectral calibration for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl Genetic Analyzers Perform a new spectral calibration and re run the samples Poor matrix for the ABI PRISM 310 Genetic Analyzer Re run and optimize the matrix Use of Classic mode analysis method Use of Classic mode analysis on samples can result in baselines with more noise than those analyzed using the Basic or Advanced mode analysis method Advanced mode analysis methods and size standards are recommended Red bar appears during analysis If none of the samples had matrices applied when run on the of samples and the following ABI PRISM 310 Genetic Analyzer no data will be displayed error message appears when data Apply a matrix file during analysis in the GeneMapper ID are displayed Some selected software and re analyze sample s do not contain analysis data Those sample s will not be shown Error message after attempting There was a conflict between different sets of panel and bin to import panel and bin files files Delete all panel and bin sets and re import files in a Unable to save panel data different order java SQLEException ORA 00001 unique constraint IFA CKP_NNN violated
85. r forensic and paternity samples 36 This novel system uses paramagnetic particles to prepare clean samples for STR analysis easily and efficiently and can be used to extract DNA from stains or liquid samples such as blood or solutions The DNA IQ Resin eliminates PCR inhibitors and contaminants frequently encountered in casework samples With larger samples the DNA IQ System delivers a consistent amount of total DNA The system has been used to isolate and quantify DNA from routine sample types including buccal swabs stains on FTA paper and liquid blood Additionally DNA has been isolated from casework samples such as tissue differentially separated sexual assault samples and stains on support materials The DNA IQ System has been tested with the PowerPlex Systems to ensure a streamlined process See Section IX I for ordering information For applications requiring human specific DNA quantification the Plexor HY System Cat DC1000 has been developed 37 See Section IX I for ordering information The DNA IQ System has been fully automated on the Beckman Coulter Biomek 2000 Laboratory Automation Workstation 38 Biomek 3000 Laboratory Automation Workstation 39 and Tecan Freedom EVO Liquid Handler 40 In addition the DNA IQ Reference Sample Kit for Maxwell 16 Cat AS1040 and DNA IQ Casework Sample Kit for Maxwell 16 are available 41 42 For information on automation of laboratory processes on aut
86. rameters can be saved in the Params folder in most installations this is located at C AppliedBio Shared Analysis Sizecaller Params 5 Apply the stored analysis parameters file to the samples 6 Assign a new size standard Select a sample file and highlight the arrow next to size standard Select define new Assign the size standard peaks as shown in Figure 13 in Section IX E Store the size standard in the Size Standards folder at C AppliedBio Shared Analysis Sizecaller SizeStandards 7 Apply the size standard file to the samples then analyze the sample files See Section VI F for additional information on the use of the PowerTyper 16 Macro Release 2 0 and Genotyper software 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 29 Page 30 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 TMD012 Printed in USA Revised 5 08 VI D Sample Analysis Using the GeneScan Software and PC Operating Systems continued Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull up
87. rument detection limits vary therefore the amount of product mixed with loading cocktail may need to be increased or decreased If peak heights are higher than desired samples can be diluted in Gold ST R 1X Buffer before mixing with loading cocktail This may result in uneven allele peak heights across loci For best results use less template DNA in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles i e 10 18 or 10 20 cycling 4 Combine 2 0 l of prepared loading cocktail and 1 0 l of PowerPlex 16 Allelic Ladder Mix Vortex the allelic ladder mix prior to pipetting 5 Briefly centrifuge samples to bring the contents to the bottom of the tubes 6 Just prior to loading the gel denature samples by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath Denature samples just prior to loading the gel 7 After the 15 to 20 minute pre run pause the instrument by selecting Pause By pausing the pre run the water will continue to circulate keeping the gel warm during sample loading 8 Use a 60cc syringe filled with buffer and fitted with a bent 18 gauge needle to flush urea from the well area 9 Load 1 5 l of each denatured sample into the respective wells You may need to optimize the volume of sample loaded for individual instruments We recommend loading volumes of 1 0 2 0 l 10 Place the lid on the upper buffer ch
88. s tmd012 0508 qxp 9 16 2008 3 48 PM Page 40 Page 41 Symptoms Causes and Comments Size standard not called Starting data point was incorrect for the partial range chosen correctly Figure 12 in Section VI B Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in advanced mode size standard Open the size match editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak threshold in the analysis method for the red channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks Error message The size standard and analysis method were not in the same Either panel size standard mode Classic vs Basic or Advanced Be sure both files or analysis method is invalid are set to the same mode either Classic or Basic or Advanced mode No alleles called but no error Panel was not selected for sample In the Panel column select message appears the appropriate panel set for the STR system t
89. s from one color to another may be observed Saturated signal may appear also as two peaks split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights also may appear less uniform 3 There can be variation between instruments regarding the relative fluorescence levels detected using the same sample Furthermore different instruments vary in the relative efficiency of color detection affecting the dye color to dye color balance 5684TA Figure 8 The Analysis Parameters window The start point of the analysis range which will vary is defined in Section VI D Step 2 tmd012 0508 qxp 9 16 2008 3 48 PM Page 30 Page 31 VI E Sample Analysis Using the GeneScan Software and Macintosh Operating Systems 1 Analyze data using the GeneScan analysis software 2 Review the raw data for one or more sample runs Highlight the sample file name then in the Sample menu select raw data Move the cursor so the crosshair is on the baseline to the right of the large primer peak before the first internal lane standard peak red Use the X value number shown at the bottom left of the window for the start position in the analysis parameters 3 The recommended analysis parameters are 4 The analysis parameters can be saved in the Params folder 5
90. s for peak amplitude thresholds are usually 50 200RFU and should be determined by individual laboratories tmd012 0508 qxp 9 16 2008 3 48 PM Page 31 Page 32 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 TMD012 Printed in USA Revised 5 08 VI E Sample Analysis Using the GeneScan Software and Macintosh Operating Systems continued Notes 1 Peak heights outside the linear range of the instrument may generate artifact peaks due to instrument saturation i e overloading the sample Bleedthrough pull ups from one color to another may be observed Saturated signal may appear also as two peaks split peak 2 If peak heights are not within the linear range of detection of the instrument the ratio of stutter peaks to real allele peaks increases and allele designations become difficult to interpret The balance of peak heights also may appear less uniform 3 There can be variation between instruments regarding the relative fluorescence levels detected using the same sample Furthermore different instruments vary in the relative efficiency of color detection affecting the dye color to dye color balance VI F Sample Analysis Using the Genotyper Software and PowerTyper 16 Macro To facilitate analysis of data generated with the PowerPlex 16 System we have created a file to allow autom
91. scrimination J Forensic Sci Soc 12 355 9 35 Brenner C and Morris J W 1990 In Proceedings from the International Symposium on Human Identification 1989 Promega Corporation 21 53 36 Mandrekar P V Krenke B E and Tereba A 2001 DNA IQ The intelligent way to purify DNA Profiles in DNA 4 3 16 37 Krenke B E et al 2005 Development of a novel fluorescent two primer approach to quantitative PCR Profiles in DNA 8 1 3 5 38 Greenspoon S and Ban J 2002 Robotic extraction of sexual assault samples using the Biomek 2000 and the DNA IQ System Profiles in DNA 5 1 3 5 39 McLaren B Bjerke M and Tereba A 2006 Automating the DNA IQ System on the Biomek 3000 Laboratory Automation Workstation Profiles in DNA 9 1 11 13 40 Cowan C 2006 The DNA IQ System on the Tecan Freedom EVO 100 Profiles in DNA 9 1 8 10 41 Bjerke M et al 2006 Forensic application of the Maxwell 16 Instrument Profiles in DNA 9 1 3 5 42 Mandrekar P et al 2007 Introduction to Maxwell 16 low elution volume configuration for forensic casework Profiles in DNA 10 2 10 12 Additional STR references can be found at www promega com geneticidentity IX Appendix IX A Advantages of STR Typing STR typing is more tolerant of degraded DNA templates than other typing methods because amplification products are less than 500bp long much smaller than material det
92. select No Selection in the Analysis Module 1 column Analysis parameters can be applied after data collection and during data analysis using the GeneScan analysis software 12 Select OK This new plate record will appear in the pending plate records table on the plate setup page of the collection software 13 Place samples in the instrument and close the instrument doors 14 Locate the pending plate record that you just created and click once on the name 15 Once the pending plate record is highlighted click on the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples to link the plate to the plate record 16 When the plate record is linked to the plate the plate graphic will change from yellow to green the plate record moves from the pending plate records table to the linked plate records table and the Run Instrument button becomes enabled 17 Select Run Instrument on the toolbar to start the sample run 18 Monitor electrophoresis by observing the run status array and capillary views windows in the collection software Each injection will take approximately 45 minutes 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 47 PM Page 15 Page 16 Promega Corpor
93. splay all data or POWER 20 Filter macro option All four dye colors were not imported For Genotyper software versions 2 5 and 3 5 or higher set preferences in the Edit menu to import the blue green yellow and red colors The Check ILS macro All four dye colors were not imported For Genotyper displays an empty plot software versions 2 5 and 3 5 or higher set preferences in the window Edit menu to import the blue green yellow and red colors Off ladder peaks Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a different injection of allelic ladder to determine sizes in the PowerTyper 16 Macro Release 2 0 Do not use the first injection on a new column for the ladder sample The base pair size of alleles was incorrect because incorrect fragment sizes were assigned to the internal lane standard Confirm that internal lane standard fragment sizes are assigned correctly Re analyze the sample using GeneScan software and redefine the internal lane standard fragments VIII References 1 Edwards A et al 1991 DNA typing with trimeric and tetrameric tandem repeats Polymorphic loci detection systems and population genetics In The Second International Symposium on Human Identification 1991 Promega Corporation 31 52 2 Edwards A et al 1991 DNA typing and genetic mapping with trimeric and tetram
94. t least one ILS 600 fragment smaller than the smallest sample peak and at least one ILS 600 fragment larger than the largest sample peak Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks defined in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Off ladder alleles An allelic ladder from a different run than the samples was used Re analyze samples with an allelic ladder from the same run The GeneMapper ID software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section VI B or VI C Panel file selected for analysis was incorrect for the STR system used Assign correct panel file that corresponds to the STR system used for amplification The allelic ladder was not identified as an allelic ladder in the sample type column The wrong analysis type was chosen for the analysis method Be sure to use the HID analysis type The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample 5685TA Figure 11 The error message that appears in the GeneMapper ID software when the analysis parameters and size standard have different analysis type
95. the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 11 B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 13 C Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer 16 D Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer 18 VI Data Analysis 22 A PowerPlex Panel and Bin Sets with GeneMapper ID Version 3 2 22 B Creating a Casework Analysis Method with GeneMapper ID Software 23 C Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software 27 D Sample Analysis Using the GeneScan Software and PC Operating Systems 29 E Sample Analysis Using the GeneScan Software and Macintosh Operating Systems
96. the toolbar to start the sample run 11 Monitor electrophoresis by observing the run view array or capillaries viewer window in the data collection software Each injection will take approximately 45 minutes V B Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath aerosol resistant pipette tips 3100 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 10X genetic analyzer buffer with EDTA MicroAmp optical 96 well plate and septa for the 3100 Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 3100 3130 Cat DG4650 The quality of formamide is critical Use Hi Di formamide with a conductivity less than 100 S cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100 S cm 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 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
97. tration or altered pH If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the DNA volume should not exceed 20 of the total reaction volume Carryover of K Na Mg2 or EDTA from the DNA sample can negatively affect PCR A change in pH may also affect PCR Store DNA in TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or nuclease free water Thermal cycler plate or tube problems Review the thermal cycling protocols in Section IV B We have not tested other reaction tubes plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Mix the 10X PowerPlex 16 Primer Pair for 15 seconds using a vortex mixer before use Poor capillary electrophoresis injection ILS 600 peaks also affected Re inject the sample Check the syringe for leakage Check the laser power Poor quality formamide was used Use only Hi Di formamide when analyzing samples 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 TMD012 Revised 5 08 tmd012 0508 qxp 9 16 2008 3 48 PM Page 37 Page 38 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 TMD012 Pr
98. ystem 8 Monitor electrophoresis by observing the raw data and status windows Each sample will take approximately 40 minutes for syringe pumping sample injection and sample electrophoresis V D Detection of Amplified Fragments Using the ABI PRISM 377 DNA Sequencer Materials to Be Supplied by the User Solution compositions are provided in Section IX H Long Ranger gel solution Cambrex Cat 50611 or Long Ranger Singel pack for ABI 377 36cm Cambrex Cat 50691 10 Ammonium Persulfate Cat V3131 TEMED Urea Cat V3171 TBE 10X buffer Nalgene tissue culture filter 0 2 micron 36cm front and rear glass plates 36cm gel spacers 0 2mm thick 36 well sharkstooth comb or 34 well square tooth comb 0 2mm thick clamps e g large office binder clamps gel loading pipette tips aerosol resistant pipette tips Section IX I Liqui Nox or other detergent PowerPlex Matrix Standards 310 Cat DG4640 Blue Dextran Loading Solution Cat DV4351 crushed ice or ice water bath 95 C dry heating block water bath or thermal cycler Caution Acrylamide Long Ranger gel solution is a neurotoxin and suspected carcinogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with acrylamide solutions tmd012 0508 qx

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