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PowerPlex® 18D System

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1. 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 TMD031 Printed in USA Revised 5 14 Page 42 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 42 6 K Controls 1 Observe the results for the negative control Using the protocols defined in this manual the negative control should be devoid of amplification products 2 Observe the results for the 2800M Control DNA Compare the 2800M DNA allelic repeat sizes with the locus specific allelic ladder The expected 2800M Control DNA allele designations for each locus are listed in Table 6 Section 9 A 6 L Results Representative results of the PowerPlex 18D System are shown in Figure 21 The PowerPlex 18D Allelic Ladder Mix is shown in Figure 22 Figure 21 The PowerPlex 18D System Two 1 2mm punches were taken from a buccal sample transferred to an FTA card and amplified for 27 cycles using the PowerPlex 18D System Amplification products were mixed with CC5 Internal Lane Standard 500 and analyzed with an Applied Biosystems 3130xl 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 loci D3S1358 TH01 D21S11 D18S51 and Penta E Panel B An electropherogram showing the peaks of the JOE labeled loci D5S818 D13S317 D7S820 D16S539
2. TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 12 PCR Optimization Cycle number should be optimized based on the results of an initial experiment to determine the sensitivity with your collection method sample types and instrumentation 1 Choose several samples that represent typical sample types you encounter in the laboratory Prepare them as you would using your normal workflow 2 Prepare three identical reaction plates with aliquots of the same swab extracts 3 Amplify samples using the thermal cycling protocol provided above but subject each plate to a different cycle number 26 27 and 28 cycles 4 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type 5 Instrument Setup and Sample Preparation 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3500 3500xL capillary array 36cm 96 well retainer amp base set standard Applied Biosystems Cat 4410228 POP 4 polymer for the Applied Biosystems 3500 or 3500xL Genetic Analyzer anode buffer container cathode buffer container conditioning reagent pouch for the Applied Bio
3. D 5X Master Mix 5 0 l PowerPlex 18D 5X Primer Pair Mix 5 0 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex D 5X Master Mix and PowerPlex 18D 5X Primer Pair Mix For FTA card punches the template DNA will be added at Step 6 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 8 Thermal Cycling This manual contains protocols for use of the PowerPlex 18D System with the GeneAmp PCR system 9700 thermal cycler Amplification and detection instrumentation may vary You will need to optimize protocols including cycle number 26 29 cycles and injection conditions for each laboratory instrument Testing at Promega shows that 27 cycles works well for a variety of sample types Buccal samples may require more amplification cycles than blood samples Cycle number will need to be optimized in each laboratory for each sample type that is amplified 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycle time is less than 1 5 hours 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts PCR Optimization Cycle number should be optimized based
4. Kline M C et al 2005 Results from the NIST 2004 DNA quantitation study J Forensic Sci 50 570 8 Additional STR references can be found at www promega com geneticidentity 9 Appendix 9 A Advantages of Using the Loci in the PowerPlex 18D System The loci included in the PowerPlex 18D System Tables 4 and 5 were 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 18D System amplifies all CODIS core loci in a single reaction The PowerPlex 18D 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 18D System a single amplification system with a power of exclusion sufficient to resolve paternity disputes definitively The Amelogenin locus is included in the PowerPlex 18D System to allow gender identification of each sample Finally D2S1338 and D19S433 were added because of their popularity and inclusion in a number of state databases within the U S Table 6 lists the PowerPlex 18D System alleles revealed in the 2800M Control DNA Terminal nucleotid
5. to save your settings 14 Select Done to exit the GeneMapper ID X Manager Processing Data for Samples with No Global Filter 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Allelic Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Allelic Ladder in the Sample Type column for proper genotyping 5 In the Analysis Method column select the analysis method created above 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 TMD031 Printed in USA Revised 5 14 Page 34 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM 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 Printed in USA Part TMD031 Revised 5 14 6 In the Panel column select the panels text file that was imported in Section 6 A 7 In the Size Standard column select the size standard that was created in S
6. 215 5 2 6 2 8 12 12 2 13 13 2 14 14 2 15 15 2 16 16 2 17 17 2 18 18 2 1The length of each allele in the allelic ladder has been confirmed by sequence analyses 2When using an internal lane standard such as the CC5 ILS 500 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 3For 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 4Amelogenin is not an STR but displays a 109 base X specific band and a 115 base Y specific band TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 61 Table 6 The PowerPlex 18D System Allele Determinations in Commonly Available Standard DNA Templates STR Locus Standard DNA Templates1 2800M 9947A 99482 D3S1358 17 18 14 15 15 17 TH01 6 9 3 8 9 3 6 9 3 D21S11 29 31 2 30 30 29 30 D18S51 16 18 15 19 15 18 Penta E 7 14 12 13 11 11 D5S818 12 12 11 11 11 13 D13S317 9 11 11 11 11 11 D7S820 8 11 10 11 11 11 D16S539 9 13 11 12 11 11 CSF1PO 12 12 10 12 10 11 12 Penta D 12 13 12 12 8 12 Amelogenin X Y X X X Y vWA 16 19 17 18 17 17 D8S1179 14 15 13 13 12 13
7. Alternatively a previously created File Name Convention may be used Select the File Name Attributes according to laboratory practices and save with a descriptive name Figure 7 The Create New File Name Convention window 9252TA 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 TMD031 Printed in USA Revised 5 14 Page 20 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 20 7 To create a new Results Group Figure 8 navigate to the Library Select Results Group then select Create Alternatively a previously created Results Groups may be used Select the Results Group Attributes according to laboratory practices Save with a descriptive name 8 To create a New Plate navigate to the Library Figure 9 and from the Manage menu select Plates then Create 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 TMD031 Revised 5 14 Page 21 Figure 8 The Create New Results Group window 9253TA Figure 9 Defining plate properties 9254TA TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 21 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 9 Assign a descript
8. CSF1PO and Penta D Panel C An electropherogram showing the peaks of the TMR ET labeled loci Amelogenin vWA D8S1179 TPOX and FGA Panel D An electropherogram showing the peaks of the CXR ET labeled loci D19S433 and D2S1338 Panel E An electropherogram showing the 60bp to 500bp fragments of the CC5 Internal Lane Standard 500 9654TA A B C D E Page 43 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 TMD031 Revised 5 14 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 43 9655TA A B C D Figure 22 The PowerPlex 18D Allelic Ladder Mix The PowerPlex 18D 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 18D panels and bins text 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 ET labeled allelic ladder components and their allele designations Panel D The CXR ET labeled allelic ladder components and their allele designations Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330
9. Direct Amplification of DNA from Storage Card Punches continued 3 Prepare four identical reaction plates with punches from the same samples 4 Amplify samples using the thermal cycling protocol provided above but subject each plate to a different cycle number 26 29 cycles 5 Following amplification use your laboratory s validated separation and detection protocols to determine the optimal cycle number for the sample type and number of storage card punches 4 B Direct Amplification of DNA from Swabs Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips SwabSolution Kit Cat DC8271 This section contains a protocol for amplifying swab extracts using the PowerPlex 18D System and GeneAmp PCR System 9700 thermal cycler Pretreat cotton swabs or OmniSwabs GE Healthcare with the SwabSolution Kit Cat DC8271 as described in the SwabSolution Kit Technical Manual TMD037 to generate a swab extract Be sure to include a blank swab as a negative control when processing samples Amplification Setup 1 Thaw the PowerPlex D 5X Master Mix and PowerPlex 18D 5X Primer Pair Mix completely Note Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 5X
10. Do not reduce the reaction volume below 25 l See Section 6 L for additional information on stutter and artifacts Artifacts of STR amplification Amplification of STRs 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 a 20 minute extension step at 60 C after thermal cycling Section 4 or 9 C Peak height imbalance Excessive amount of DNA Amplification of gt 20ng of template can result in an imbalance with smaller loci showing more product than larger loci Use one or two 1 2mm punches from a storage card containing a buccal sample or one 1 2mm punch from a storage card containing whole blood Follow the manufacturer s recommendations when depositing sample onto the storage card Decrease number of cycles The reaction volume was too low This system is optimized for a final reaction volume of 25 l to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume can result in suboptimal performance Amplification was inhibited when using more than one storage card punch with blood Use only one 1 2mm storage card punch with blood 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 TMD031 Printed in USA Revised 5 14 Page 50 TMD031 0514 TMD031 0514 5 28 2014 1
11. Part TMD031 Printed in USA Revised 5 14 7 Troubleshooting 46 A Amplification and Fragment Detection 46 B Direct Amplification of DNA from Storage Card Punches 49 C Direct Amplification of DNA from Swabs 52 D GeneMapper ID X Software 54 E GeneMapper ID Software 55 8 References 58 9 Appendix 59 A Advantages of Using the Loci in the PowerPlex 18D System 59 B The CC5 Internal Lane Standard 500 63 C Protocol for Amplification of Extracted DNA 64 D Composition of Buffers and Solutions 66 E
12. Part TMD031 Printed in USA Revised 5 14 Page 14 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 14 Instrument Preparation Refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide for the instrument maintenance schedule and instructions to install the capillary array buffers and polymer pouch and perform a spatial calibration Samples may be analyzed as described in the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 1 Open the 3500 Data Collection Software The Dashboard screen will launch Figure 2 Ensure that the Consumables Information and Maintenance Notifications are acceptable Set the oven temperature to 60 C then select Start Pre Heat at least 30 minutes prior to the first injection to preheat the oven 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 TMD031 Revised 5 14 Page 15 Figure 2 The Dashboard 9247TA TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 15 Page 16 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 TMD031 Printed in USA Revised 5 14 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 2 To create a new Instrume
13. 1ml PowerPlex 18D 5X Primer Pair Mix 25 l 2800M Control DNA 10ng l 10 1 250 l Water Amplification Grade Post amplification Components Box 4 50 l PowerPlex 18D Allelic Ladder Mix 8 150 l CC5 Internal Lane Standard 500 The PowerPlex 18D Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post amplification box after opening The Water Amplification Grade is provided in a separate sealed bag for shipping This component should be moved to the pre amplification box after opening Storage Conditions For long term storage store all components except the 2800M Control DNA at 30 C to 10 C in a nonfrost free freezer Store the 2800M Control DNA at 2 10 C The PowerPlex 18D System components can be stored for up to 1 week at 2 10 C The PowerPlex 18D 5X Primer Pair Mix PowerPlex 18D Allelic Ladder Mix and CC5 Internal Lane Standard 500 CC5 ILS 500 are light sensitive and must be stored in the dark We strongly recommend that pre amplification and post amplification reagents be stored and used separately with different pipettes tube racks etc Available Separately The proper panels and bins text files for use with GeneMapper ID and ID X software are available for download at www promega com resources tools genemapper id software panels and bin sets Matrix standards are required for initial setup of the color separation matrix
14. Fax 608 277 2516 www promega com Part TMD031 Printed in USA Revised 5 14 Page 44 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 44 6 L Results continued Artifacts and Stutter Stutter products are a common amplification artifact associated with STR analysis 13 14 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 The number of stutter peaks can increase with some loci such as D3S1358 for samples that are particularly intense high signal level or template amount 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 mean stutter plus three standard deviations at each locus is used in the PowerPlex 18D panels text files for locus specific filtering in the GeneMapper ID software version 3 2 and GeneMapper ID X software In addition to stutter peaks other artifact peaks can be observed at some of the PowerPlex 18D System loci Table 3 Low level products can be seen in the n 2 and n 2 positions with some loci such as D21S11 and D5S818 Artifact peaks may be seen in the D5S818 and D7S820 regions of the JOE channel These extra peaks occur when the amplified peaks are particularly intense formamide polymer or capillary was of poor quality or denatu
15. Flags tab You may change these settings 13 Select OK to save your settings 9649TA Figure 19 The GeneMapper ID Peak Detector tab 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 TMD031 Revised 5 14 Page 39 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 39 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 TMD031 Printed in USA Revised 5 14 Page 40 6 I Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 continued Processing Data for Databasing or Paternity Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Ladder in the Sample Type column for proper genotyping 5 In the Analysis Method column select the analysis method created previous
16. PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers The PowerPlex 5 Dye Matrix Standards 310 cannot be used to generate a matrix on these instruments For protocols and additional information about spectral calibration see the PowerPlex 5 Dye Matrix Standards 3100 3130 Technical Bulletin TBD024 This manual is available online at www promega com protocols Page 5 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 5 4 Protocols for Direct Amplification Using the PowerPlex 18D System The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre amplification and post amplification 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 7 The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm Quantification of this control DNA by other methods such as qPCR may result in a different value Prepare a fresh DNA dilution for each set of amplifications Do not store diluted DNA e g 0 25ng l or less A protocol to amplify extracted DNA is provided in Section 9 C 4 A Direct Amplification of DNA from Storage Card Punches Materials
17. Reagent into amplification reaction We recommend treating one 1 2mm nonFTA card punch with 10 l of PunchSolution Reagent and using one punch per 25 l amplification reaction Use of a smaller amplification reaction volume may compromise performance if using 10 l of PunchSolution Reagent Reducing the PunchSolution Reagent volume may improve results when using a reduced amplification reaction volume Laboratory optimization and validation are required Extreme variability in sample There can be significant individual to individual variability in to sample peak heights the deposition of cells onto a punch resulting in peak height variability between samples The PunchSolution Kit increases the recovery of amplifiable DNA from samples but does not normalize the amount of DNA present Page 51 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 51 7 C Direct Amplification of DNA from Swabs The following information is specific to direct amplification of DNA from swabs For information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refrigerator door whe
18. Requirement Summary in the Options menu and correct the necessary analysis requirements to continue analysis Edits in label edit viewer cannot To view edits made to a project the project first must be be viewed saved Close the plot view window return to the main GeneMapper ID X page and save the project Display the plot window again then view the label edit table Marker header bars for some loci When an edit is made to a locus the quality flags and marker are gray header bar automatically change to gray To change the GQ and marker header bar for a locus to green override the GQ in the plot window Alleles not called To analyze samples with GeneMapper ID X software at least one allelic ladder must be defined An insufficient number of CC5 ILS 500 fragments were defined Be sure to define at least one CC5 ILS 500 fragment smaller than the smallest sample peak or allelic ladder peak and at least one CC5 ILS 500 fragment larger than the largest sample peak or allelic ladder peak In this instance the allelic ladder would have failed the allelic ladder quality check Run was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 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 A low quality allelic ladder was used during analysis Ensure that only high qua
19. TPOX 11 11 8 8 8 9 FGA 20 23 23 24 24 26 D19S433 13 14 14 15 13 14 D2S1338 22 25 19 23 23 23 1Information on strains 9947A and 9948 is available online at http ccr coriell org Sections Collections NIGMS SsId 8 Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 23 2Strain 9948 displays three alleles at the CSF1PO locus The peak height for allele 12 is much lower than those for alleles 10 and 11 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 TMD031 Printed in USA Revised 5 14 Page 62 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 62 9 B The CC5 Internal Lane Standard 500 The CC5 Internal Lane Standard 500 contains 21 DNA fragments of 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases in length Figure 23 Each fragment is labeled with CC5 dye and can be detected separately as a fifth color in the presence of PowerPlex 18D amplified material The CC5 ILS 500 is designed for use in each CE injection to increase precision in analyses when using the PowerPlex 18D System Protocols to prepare and use this internal lane standard are provided in Section 5 Note Sizing of alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be
20. The matrix standards are provided separately and are available for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers PowerPlex 5 Dye Matrix Standards 3100 3130 Cat DG4700 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 TMD031 Printed in USA Revised 5 14 Page 4 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM 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 Printed in USA Part TMD031 Revised 5 14 3 Before You Begin 3 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 10 11 Guidelines for the validation process are published in the Internal Validation of STR Systems Reference Manual 12 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 Additional research and validation are required if any modifications to the recommended protocols are made PCR based STR analysis is subj
21. and 3130xl Genetic Analyzers lengthen the run time in the Run Module in the Module Manager Step 5 B Step 1 For the 3500 and 3500xL Genetic Analyzers lengthen the run time in the Instrument Protocol window Step 5 A Step 2 Otherwise changes to the Instrument Protocol such as lengthening the run time made in the Assay window may not be incorporated into the instrument run 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 TMD031 Printed in USA Revised 5 14 Page 46 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM 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 Printed in USA Part TMD031 Revised 5 14 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 denatured completely Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to capillary electrophoresis Do not cool samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Artifacts of STR amplification Amplification
22. labeled as OL 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 TMD031 Revised 5 14 Page 63 Figure 23 CC5 Internal Lane Standard 500 An electropherogram showing the CC5 Internal Lane Standard 500 fragments 8248TA TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 63 9 C Protocol for Amplification of Extracted DNA Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate or 0 2ml MicroAmp reaction tubes Applied Biosystems aerosol resistant pipette tips Testing at Promega has shown successful amplification of 1ng of template DNA in a 25 l reaction volume using the protocols detailed below Alternatively 5ng of template DNA can be used but the number of cycles should be decreased to 27 cycles Cycle number and quantity of input DNA can be optimized to achieve the desired sensitivity and signal Protocols for direct amplification using the PowerPlex 18D System are provided in Section 4 The locus to locus balance for the PowerPlex 18D System is optimized for direct amplification methods Amplification of extracted DNA may result in locus to locus imbalance The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contami
23. need to optimize these settings In house validation should be performed 9648TA Figure 13 The GeneMapper ID X Allele tab 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 TMD031 Printed in USA Revised 5 14 Page 30 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 30 10 Select the Peak Detector tab Figure 14 shows an example of settings used at Promega You may need to optimize these settings In house validation should be performed Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU for data generated on the 3130 series instruments and 175RFU and higher for data generated on the Applied Biosystems 3500 and 3500xL Genetic Analyzers Individual laboratories should determine their peak amplitude thresholds from internal validation studies Peak heights for the CC5 ILS are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower
24. number of cycles should be decreased to 27 cycles TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 65 9 C Protocol for Amplification of Extracted DNA continued Thermal Cycling Amplification and detection instrumentation may vary You will need to optimize protocols including cycle number 26 30 cycles and injection conditions or loading volume for each laboratory instrument Testing at Promega shows that 30 cycles works well for 1ng of DNA You may need to optimize cycle number 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycle time is less than 1 5 hours 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts 9 D Composition of Buffers and Solutions Thermal Cycling Protocol1 96 C for 2 minutes then 94 C for 10 seconds 60 C for 1 minute for 30 cycles then 60 C for 20 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with 9600 as the ramp speed The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select 9600 for the ramp speed and enter the reaction volume Promega
25. of STRs 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 20 minute extension step at 60 C after thermal cycling Section 4 or 9 C Decrease the number of cycles Plasticware can alter heat transfer during amplification and prevent full adenylation Increase the final extension time Artifacts The signal strength of certain artifacts increases with storage of the amplification plate at 4 C see Table 5 sometimes in as short a time period as overnight but more commonly when left at 4 C for a few days We recommend storing amplification products at 20 C Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis Appearance of shadow peaks migrating in front of the main peaks especially if the shadow peaks are separated by the same distance as the main peaks in a heterozygote can indicate the presence of double stranded DNA due to incomplete denaturation or post injection re annealing 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 samples to confirm Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See inst
26. on the results of an initial experiment to determine the sensitivity with your collection method sample types number of punches and instrumentation 1 Choose several samples that represent the typical sample types you encounter in the laboratory Prepare them as you would using your normal workflow 2 Depending on your preferred protocol place one or two 1 2mm storage card punches containing a buccal sample or one 1 2mm punch of a storage card containing whole blood in each well of a reaction plate Thermal Cycling Protocol1 96 C for 2 minutes then 94 C for 10 seconds 60 C for 1 minute for 27 cycles then 60 C for 20 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with 9600 as the ramp speed The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select 9600 for the ramp speed and enter the reaction volume 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 TMD031 Revised 5 14 Page 9 TMD031 0514 TMD031 0514 5 28 2014 12 18 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 TMD031 Printed in USA Revised 5 14 4 A
27. spectral calibration and re run the samples 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 Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See instrument preparation instructions in Section 5 B Step 6 Error message after attempting There was a conflict between different sets of panels and bins to import panel and bin files text files Check to be sure that the bins are installed properly Unable to save panel data If not delete all panels and bins text files and re import files java SQLEException in a different order ORA 00001 unique constraint IFA CKP_NNN violated Allelic ladder peaks 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
28. than that for other dyes 3 The normalization box can be checked regardless of whether normalization was or was not applied during data collection 9652TA Figure 14 The GeneMapper ID X Peak Detector tab 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 TMD031 Revised 5 14 Page 31 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 31 6 D Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 continued 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID X user s manual for more information 12 Select the SQ amp GQ Settings tab You may change these settings 13 Select Save to save the new analysis method 14 Select Done to exit the GeneMapper ID X Manager Processing Data for Databasing or Paternity Samples 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of run files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Allelic Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the proje
29. than that for the other dyes 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 change these settings 13 Select OK to save your settings Processing Data for Samples with No Global Filter 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to the location of the run files Highlight desired files then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Ladder in the Sample Type column for proper genotyping 5 In the Analysis Method column select the analysis method created previously in this section 6 In the Panel column select the panels text file that was imported in Section 6 F 7 In the Size Standard column select the size standard that was created in Section 6 G or imported in Section 6 H 8 Select Analyze green arrow button to start data analysis Note Sizing of alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL Promega Corporation
30. the PowerPlex 18D System on the Applied Biosystems 3500 or 3500xL Genetic Analyzer Figure 5 shows one option for these settings Figure 5 The Create New QC Protocol window 9228TA 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 TMD031 Printed in USA Revised 5 14 Page 18 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 18 5 To create a new Assay navigate to the Library Select Assays then select Create Alternatively a previously created Assay may be used In the Create New Assay window Figure 6 select the instrument protocol created in Step 2 and the QC protocol created in Step 4 Assign a descriptive assay name Select the application type HID An Assay is required for all named samples on a plate 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 TMD031 Revised 5 14 Page 19 Figure 6 The Create New Assay window 9229TA TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 19 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 6 To create a new File Name Convention Figure 7 navigate to the Library Select File Name Conventions then select Create
31. the injection time is 5 seconds the injection voltage is 3kV and the run time is 1 500 seconds Give a descriptive name to your run module and select OK 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 G5 in the dye set drop down list Select OK The recommended final run module settings are 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 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 in
32. vary You will need to optimize protocols including cycle number 26 28 cycles and injection conditions or loading volume for each laboratory instrument Testing at Promega shows that 27 cycles works well for a variety of sample types Cycle number will need to be optimized in each laboratory for each sample type that is amplified 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycle time is approximately 1 hour 3 After completion of the thermal cycling protocol store amplified samples at 20 C in a light protected box Note Long term storage of amplified samples at 4 C or higher may produce artifacts 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 TMD031 Printed in USA Revised 5 14 Page 12 Thermal Cycling Protocol1 96 C for 2 minutes then 94 C for 10 seconds 60 C for 1 minute for 27 cycles then 60 C for 20 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with 9600 as the ramp speed The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select 9600 for the ramp speed and enter the reaction volume
33. vortexing as this may cause the reagents to be concentrated at the bottom of the tube 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 consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 Use a clean 0 2ml MicroAmp plates for reaction assembly and label appropriately 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 TMD031 Revised 5 14 Page 7 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 7 4 A Direct Amplification of DNA from Storage Card Punches continued 4 Add the final volume of each reagent listed in Table 1 to a sterile tube 5 Vortex the PCR amplification mix for 5 10 seconds then pipet 25 l of PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance Note For nonFTA card punches add the PCR amplification mix to the pretreated punches For FTA card punches add the storage card punch in Step 6 It also is acceptable to add the FTA card punch first then ad
34. 08 274 4330 Fax 608 277 2516 www promega com Part TMD031 Printed in USA Revised 5 14 Page 32 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 32 1 Select Tools then GeneMapper ID X Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 In the Analysis Method Editor window select GeneMapper ID X Security Group as the Security Group This allows access for all users of the software Other security groups may be used 5 Enter a descriptive name for the analysis method such as PowerPlex18D 6 Select the Allele tab Figure 15 7 Select the bins text file that was imported in Section 6 A 8 Ensure that the Use marker specific stutter ratio if available box is checked 9 We recommend the values shown in Figure 15 for proper filtering of stutter peaks when using the PowerPlex 18D System You may need to optimize these settings In house validation should be performed 9653TA Figure 15 The GeneMapper ID X Allele tab 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 TMD031 Revised 5 14 Page 33 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 33 6 E Creating a No Global Filter Analysis Method with GeneMapper ID X Software Version 1 2 continued 10
35. 2 18 PM 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 Printed in USA Part TMD031 Revised 5 14 Symptoms Causes and Comments Peak height imbalance continued Bode Buccal DNA Collector devices were used without a lysis step For buccal samples on Bode Buccal DNA Collector devices we recommend pretreatment with the PunchSolution Reagent to lyse samples before adding the amplification mix Active PunchSolution Reagent carried over into the amplification reaction Larger loci are most susceptible to carryover and will drop out before the smaller loci Ensure that the heat block was set at 70 C and samples were incubated for 30 minutes Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution Reagent Using a smaller amplification reaction volume may compromise performance when using 10 l of PunchSolution Reagent Reducing the PunchSolution Reagent volume may improve results for reactions with reduced amplification volumes Optimization and validation are required Inactive PunchSolution Reagent Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze avoid multiple freeze thaw cycles as this may reduce activity Carryover of excess PunchSolution
36. 3500 manual to edit a library entry Assign a descriptive protocol name Note For more detailed information refer to the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide 3 To create a new Size Standard for the QC protocol navigate to the Library Select Size Standards then select Create Alternatively a previously created Size Standard may be used Assign the size standard the name PPlex_ILS500 or another appropriate name Choose Orange as the Dye Color The fragments in the size standard are 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Figure 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 Printed in USA Part TMD031 Revised 5 14 Figure 4 The Create New Size Standard window 9227TA TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 17 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued 4 To create a new QC Protocol navigate to the Library Select QC Protocols then select Create Alternatively a previously created QC Protocol may be used Assign a descriptive protocol name Select the size standard created in Step 3 The settings for the QC protocol should be based on the internally validated conditions for
37. 3711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TMD031 Printed in USA Revised 5 14 Page 64 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 64 5 Vortex the PCR amplification mix for 5 10 seconds then pipet PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance 6 Add the template DNA 1 0ng for each sample to the respective well containing PCR amplification mix 7 For the positive amplification control vortex the tube of 2800M Control DNA then dilute an aliquot to 1 0ng in the desired template DNA volume Add 1 0ng of the diluted DNA to a reaction well containing PCR amplification mix 8 For the negative amplification control pipet Water Amplification Grade or TE 4 buffer instead of template DNA into a reaction tube containing PCR amplification mix 9 Seal the plate Optional Briefly centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles 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 TMD031 Revised 5 14 Page 65 Table 7 PCR Amplification Mix for Amplification of Extracted DNA PCR Amplification Mix Component1 Volume Per Reaction Numbe
38. 4 TMD031 0514 5 28 2014 12 18 PM 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 Printed in USA Part TMD031 Revised 5 14 7 B Direct Amplification of DNA from Storage Card Punches The following information is specific to direct amplification of DNA from storage card punches For information about general amplification and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks The reaction volume was too low This system is optimized for a final reaction volume of 25 l to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume may result in suboptimal performance especially when amplifying DNA on storage card punches directly Poor sample deposition Shedding and collection of donor cells was variable Increase cycle number Poor sample transfer to storage card or variable sampling from the storage card Take punches from a different portion of the card Increasing cycle number also can improve low peak heights Too much sample in the reaction Use one or two 1 2mm storage card punches Follow the manufacturer s recommendations when depositing sample onto the storage card With storage cards reducing the reaction volumes below 25 l may result in amplification failure Amplification was inhibited when using more than one stor
39. 7 2516 www promega com Part TMD031 Printed in USA Revised 5 14 Page 60 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 60 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 TMD031 Revised 5 14 Page 61 Table 5 The PowerPlex 18D System Allelic Ladder Information STR Locus Label Size Range of Allelic Ladder Components1 2 bases Repeat Numbers of Allelic Ladder Components3 Penta E FL 379 474 5 24 D18S51 FL 286 366 7 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 152 195 3 9 9 3 10 11 13 3 D3S1358 FL 103 147 9 20 FGA TMR ET 314 460 14 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 32 2 33 2 42 2 43 2 44 2 45 2 46 2 48 2 50 2 TPOX TMR ET 265 293 6 13 D8S1179 TMR ET 203 251 7 19 vWA TMR ET 127 183 10 24 Amelogenin4 TMR ET 109 115 X Y Penta D JOE 376 449 2 2 3 2 5 17 CSF1PO JOE 321 357 6 15 D16S539 JOE 264 304 5 8 15 D7S820 JOE 218 250 6 14 D13S317 JOE 176 208 7 15 D5S818 JOE 122 158 7 16 D2S1138 CXR ET 223 295 10 12 14 28 D19S443 CXR ET 163
40. 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 TMD031 Printed in USA Revised 5 14 Page 66 TE 4 buffer 10mM Tris HCl 0 1mM EDTA pH 8 0 1 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 TE 4 buffer with 20 g ml glycogen 1 21g Tris base 0 037g EDTA Na2EDTA 2H2O 20 g ml glycogen Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Add glycogen Bring the final volume to 1 liter with deionized water TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 66 9 E Related Products Fluorescent STR Systems Product Size Cat PowerPlex ESX 16 System 100 reactions DC6711 400 reactions DC6710 PowerPlex ESX 17 System 100 reactions DC6721 400 reactions DC6720 PowerPlex ESI 16 System 100 reactions DC6771 400 reactions DC6770 PowerPlex ESI 17 Pro System 100 reactions DC7781 400 reactions DC7780 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 16 HS System 100 reactions DC2101 400 reactions DC2100 Po
41. Nos 0743987 and 0851867 Japan Pat No 3066984 Liechtenstein Pat Nos 0743987 and 0851867 Netherlands Pat Nos 0743987 and 0851867 Spain Pat Nos 2197193 and 2173310 Sweden Pat Nos 0743987 and 0851867 Switzerland Pat Nos 0743987 and 0851867 United Kingdom Pat Nos 0743987 and 0851867 U S Pat Nos 5 654 419 5 688 648 5 869 255 6 177 247 5 707 804 6 028 190 6 544 744 7 015 000 and 5 728 528 and other pending and foreign patent applications End User Terms and Conditions Acceptance These terms and conditions shall govern the purchase use transfer and acceptance of the products described in the purchase order quotation or invoice which products are sold and distributed by Promega to the buyer transferee of such products the End User The transfer sale of products to the End User is expressly conditional upon End User s acceptance of these terms and conditions Restrictions on Use End Users are specifically not authorized to and are forbidden from reselling transferring or distributing any products either as a stand alone product or as a component of another product The right to use the products does not in and of itself include or carry any right of the End User to any GE Healthcare Bio Sciences Corp s technology or intellectual property other than expressly provided herein End Users may not use sequence s in an attempt to reverse engineer parameters of any of GE Healthcare Bio Sciences Corp proprietary products
42. Nos 2 118 048 and 2 251 793 Korean Pat No 290332 Singapore Pat No 57050 Japanese Pat Nos 3602142 and 4034293 Chinese Pat Nos ZL99813729 4 and ZL97194967 0 European Pat No 0960207 and other patents pending c U S Pat No 6 238 863 Chinese Pat No ZL99802696 4 European Pat No 1058727 Japanese Pat No 4494630 and other patents pending d 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 e Allele sequences for one or more of the loci vWA FGA D8S1179 D21S11 and D18S51 in allelic ladder mixtures is licensed under U S Pat Nos 7 087 380 and 7 645 580 Australia Pat No 2003200444 and corresponding patent claims outside the US f TMR ET CXR ET and CC5 dyes are proprietary 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 TMD031 Printed in USA Revised 5 14 Page 68 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 68 g This product or portions thereof is manufactured and sold under license from GE Healthcare under Australia Pat No 692230 Austria Pat No E236994 Belgium Pat No 0743987 Canada Pat No 2231475 EP Pat Nos 0743987 and 0851867 France Pat Nos 0743987 and 0851867 Germany Pat Nos 19581489 69530286 8 and 0851867 Italy Pat
43. Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 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 consume a small amount of each reagent it ensures that you will have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 10 Page 11 4 Add the final volume of each reagent listed in Table 2 to a sterile tube 5 Vortex the PCR amplification mix for 5 10 seconds then pipet 23 l of PCR amplification mix into each reaction well Failure to vortex the PCR amplification mix sufficiently can result in poor amplification or locus to locus imbalance 6 Pipet 2 0 l of swab extract for each sample into the appropriate well of the reaction plate 7 For the positive amplification control vortex the tube of 2800M Control DNA then dilute an aliquot to 2 5ng l and add 2 l to a reaction well containing 23 l of PCR amplification mix Note Optimization of the amount of 2800M Control DNA may be required depending on thermal cycling conditions and laboratory preferences 8 For the negative amplifica
44. Related Products 67 F Summary of Changes 67 1 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 9 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 fluorescence detection following electrophoretic separation The PowerPlex 18D System a g is used for human identification applications including forensic analysis relationship testing and research use The system allows co amplification and four color fluorescent detection of eighteen loci seventeen STR loci and Amelogenin including D3S1358 TH01 D21S11 D18S51 Penta E D5S818 D13S317 D7S820 D16S539 CSF1PO Penta D Amelogenin vWA D8S1179 TPOX FGA D19S433 and D2S1338 The internal lane standard is labeled with a fifth dye CC5 The PowerPlex 18D System is compatible with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers Amplific
45. Revised 5 14 TMD031 PowerPlex 18D System Instruc ons for use of Products DC1802 AND DC1808 T E C H N I C A L M A N U A L TMD031 0514 TMCover indd 1 TMD031 0514 TMCover indd 1 5 28 2014 12 12 34 PM 5 28 2014 12 12 34 PM PowerPlex 18D System All technical literature is available on the Internet at www promega com protocols Please visit the web site to verify that you are using 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 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 TMD031 Revised 5 14 1 Description 2 2 Product Components and Storage Conditions 3 3 Before You Begin 5 A Precautions 5 B Spectral Calibration 5 4 Protocols fo
46. Select the Peak Detector tab Figure 14 shows an example of settings used at Promega You may need to optimize these settings In house validation should be performed Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU for data generated on the 3130 series instruments and 175RFU and higher for data generated on the Applied Biosystems 3500 and 3500xL Genetic Analyzers Individual laboratories should determine their peak amplitude thresholds from internal validation studies Peak heights for the CC5 ILS are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 3 The normalization box can be checked regardless of whether normalization was or was not applied during data collection 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 11 and 12 see the GeneMapper ID X user s manual for more information 12 Select the Quality Flags tab You may change these settings 13 Select OK
47. age card punch with blood Use only one 1 2mm storage card punch with blood Active PunchSolution Reagent carried over into the amplification reaction when using nonFTA card punches Ensure that the heat block was set at 70 C and samples were incubated for 30 minutes Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution Reagent We have not tested longer incubation times Inactive PunchSolution Reagent Thaw the PunchSolution Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can fluctuate Do not refreeze avoid multiple freeze thaw cycles as this may reduce activity Faint or absent peaks for the If the positive control reaction failed to amplify check to positive control reaction make sure that the correct amount of 2800M Control DNA was added to the reaction We recommend 5ng of 2800M Control DNA per 25 l amplification reaction Do not include a blank punch in the positive control reaction Presence of a blank punch may inhibit amplification of 2800M Control DNA Optimize the amount of 2800M Control DNA for your thermal cycling conditions and laboratory preferences Improper storage of the 2800M Control DNA Page 49 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 49 7 B Direct Amplification of DNA from Storage Card Punches continued Symptoms Causes and Comments Extra peaks visible in one or Punch was contaminated Take
48. ame mode either Classic or Basic or Advanced mode TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 56 Page 57 Symptoms Causes and Comments No alleles called but no error Panels text file was not selected for sample In the Panel message appears column select the appropriate panels text file for the STR system that was used No size standard was selected In the Size Standard 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 Error message The bins text file assigned to the analysis method was deleted Both the Bin Set used in the In the GeneMapper Manager select the Analysis Methods Analysis Method and the Panel tab and open the analysis method of interest Select the Allele must belong to the same tab and select an appropriate bins text file Chemistry Kit The wrong bins text file was chosen in the analysis method Allele tab Be sure to choose the appropriate bins text file as shown in Figure 20 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
49. ard tab 3 Select Import 4 Browse to the location of the CC5_ILS_500 xml file 5 Highlight the file then select Import 6 Select Done to save changes and exit the GeneMapper Manager 8199TA Figure 17 The Size Standard Editor 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 TMD031 Revised 5 14 Page 37 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 37 6 I Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 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 GeneMapper ID software Contact Applied Biosystems 5 Enter a descriptive name for the analysis method such as PowerPlex18D_20 filter 6 Select the Allele tab Figure 18 7 Select the bins text file that was imported in Section 6 F 8 Ensure that the Use marker specific stutter ratio if available box is checked 9 Enter the values shown in Figure 18 for proper filtering of peaks when using the PowerPlex 18D System For an explanation of the proper usage and effect of these settings refer to the Applied Biosy
50. ation and detection instrumentation may vary You may need to optimize protocols including cycle number and injection conditions or loading volume for each laboratory instrument In house validation should be performed The PowerPlex 18D System provides all materials necessary to amplify STR regions of human genomic DNA including a hot start nonproofreading thermostable DNA polymerase The PowerPlex 18D System is optimized for analysis of common database samples such as unwashed FTA card punches i e direct amplification Additionally thermal cycling time was reduced from previous PowerPlex protocols This manual contains protocols for use of the PowerPlex 18D System with the GeneAmp PCR System 9700 thermal cycler in addition to protocols to separate amplified products and detect separated material Figure 1 Protocols to operate the fluorescence detection instruments should be obtained from the instrument manufacturer TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 2 Information about other Promega fluorescent STR systems is available upon request from Promega or online at www promega com 2 Product Components and Storage Conditions Product Size Cat PowerPlex 18D System 200 reactions DC1802 Not For Medical Diagnostic Use This system contains sufficient reagents for 200 reactions of 25 l each Includes Pre amplification Components Box 1ml PowerPlex D 5X Master Mix 1ml PowerPlex 18D 5X Pr
51. ator door where the temperature can fluctuate Do not re freeze avoid multiple freeze thaw cycles as this may reduce activity Extreme variability in sample There can be significant individual to individual variability in to sample peak heights cell deposition onto buccal swabs This will appear as variability in peak heights between swab extracts The extraction process maximizes recovery of amplifiable DNA from buccal swabs but does not normalize the amount of DNA present If variability is extreme quantitate the DNA using a fluorescence based double stranded DNA quantitation method or qPCR based quantitation method The quantitation values can be used to normalize input template amounts to minimize variation in signal intensity Page 53 TMD031 0514 TMD031 0514 5 28 2014 12 18 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 TMD031 Printed in USA Revised 5 14 7 D GeneMapper ID X Software Symptoms Causes and Comments Stutter peaks not filtered Stutter file was not imported into the Panel Manager when the panels and bins text files were imported Stutter distance was not defined in the Analysis Method Allele tab Samples in the project not analyzed The Analysis Requirement Summary window was not active and there was an analysis requirement that was not met Turn on Analysis
52. blank swab as a or all color channels negative control when processing samples Artifacts of STR amplification Amplification of swab extracts with high concentrations of DNA can result in artifact peaks due to overamplification resulting in saturated signal on the CE instrument We recommend 2 l of swab extract per 25 l reaction Using more than 2 l in a 25 l reaction or using 2 l with a smaller reaction volume may result in overamplification and signal saturation If signal is saturated repeat the amplification with less swab extract or a reduced cycle number 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 TMD031 Printed in USA Revised 5 14 Page 52 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM 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 Printed in USA Part TMD031 Revised 5 14 Symptoms Causes and Comments Extra peaks visible in one Amplification of excess template for a given cycle number or all color channels continued resulted in overloading of the capillary upon electrokinetic injection In addition to signal saturation excess DNA in the capillary is difficult to maintain in a denatured single stranded state Some single stranded DNA renatures and becomes do
53. ct must contain at least one allelic ladder injection that is designated as Allelic Ladder in the Sample Type column for proper genotyping In the Analysis Method column select the analysis method created above 5 In the Panel column select the panels text file that was imported in Section 6 A 6 In the Size Standard column select the size standard that was created in Section 6 B or imported in Section 6 C 7 Select Analyze green arrow button to start data analysis Note By default the software displays the Analysis Requirement Summary Allelic Ladder Analysis Summary and Analysis Summary windows after quality review by the software Ensure that all requirements are met as each window appears If you do not have the Analysis Requirement Summary window activated you may need to do additional manual troubleshooting Note Sizing of alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL 6 E Creating a No Global Filter Analysis Method with GeneMapper ID X Software Version 1 2 These instructions are intended as a guide to start analyzing data in GeneMapper ID X Software They are not intended as a comprehensive guide for using GeneMapper ID X Software We recommend that users contact Applied Biosystems for training on the software Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 6
54. d the PCR amplification mix 6 For FTA storage cards add one or two 1 2mm punches from a card containing a buccal sample or one 1 2mm punch from a card containing whole blood to the appropriate wells of the reaction plate 7 For the positive amplification control vortex the tube of 2800M Control DNA then dilute an aliquot to 5ng l Pipet 1 l of diluted DNA into a reaction well containing 25 l of PCR amplification mix Notes 1 Do not include blank storage card punches in the positive control reactions 2 Optimization of the amount of control DNA may be required based on cycling conditions and laboratory preferences 8 Reserve a well containing PCR amplification mix as a negative amplification control Note An additional negative control with a blank punch may be performed to detect contamination from the storage card or punch device 9 Seal the plate and briefly centrifuge the plate to bring storage card punches to the bottom of the wells 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 TMD031 Printed in USA Revised 5 14 Page 8 Table 1 PCR Amplification Mix for Direct Amplification of DNA from Storage Card Punches PCR Amplification Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Amplification Grade 15 l PowerPlex
55. de addition by Taq DNA polymerase Genome Res 5 312 7 16 Magnuson V L et al 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase Implications for PCR based genotyping and cloning BioTechniques 21 700 9 17 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 18 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 19 Butler J M 2006 Genetics and genomics of core STR loci used in human identity testing J Forensic Sci 51 253 65 20 Hill C R et al 2008 Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples J Forensic Sci 53 73 80 21 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 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 58 Page 59 22 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 23 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 24
56. e addition 15 16 occurs when nonproofreading thermostable DNA polymerase adds a nucleotide generally adenine to the 3 ends of amplified DNA fragments in a template independent manner The efficiency with 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 20 minutes 17 to the amplification protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used 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 TMD031 Revised 5 14 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 59 Table 4 The PowerPlex 18D System Locus Specific Information STR Locus Label Chromosomal Location1 GenBank Locus and Locus Definition Repeat Sequence2 5 3 Penta E FL 15q NA AAAGA D18S51 FL 18q21 3 HUMUT574 AGAA 18 D21S11 FL 21q11 21q21 HUMD21LOC TCTA Complex 18 TH01 FL 11p15 5 HUMTH01 human tyrosine hydroxylase gene AATG 18 D3S1358 FL 3p NA TCTA Complex FGA TMR ET 4q28 HUMFIBRA human fibrinogen alpha chain gene TTTC Complex 18 TPOX TMR ET 2p24 2pter HUMTPOX human thyroid pe
57. e software Note The panels bins and stutter text files mentioned here are compatible with earlier versions of the GeneMapper ID X software Getting Started 1 To obtain the proper panels bins and stutter text files for the PowerPlex 18D System go to www promega com geneticidtools panels_bins 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 TMD031 Printed in USA Revised 5 14 Page 26 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 26 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 TMD031 Revised 5 14 2 Enter your contact information and select GeneMapper ID X Select Submit 3 Save the PowerPlex_18D_Panels_IDX_vX x txt PowerPlex_18D_Bins_IDX_vX x txt and PowerPlex_18D_Stutter_IDX_vX x txt files where X x refers to the most recent version of the panels bins and stutter text files to a known location on your computer Importing Panels Bins and Stutter Text Files 1 Open the GeneMapper ID X software 2 Select Tools then Panel Manager 3 Highlight the Panel Manager icon in the upper left navigation pane 4 Select File then Import Panels 5 Navigate to the panels text file downl
58. ect to contamination by very small amounts of human DNA Extreme care should be taken to avoid cross contamination when preparing template DNA handling primer pairs assembling amplification reactions and analyzing amplification products Reagents and materials used prior to amplification PowerPlex D 5X Master Mix PowerPlex 18D 5X Primer Pair Mix 2800M Control DNA and Water Amplification Grade are provided in a separate box and should be stored separately from those used following amplification PowerPlex 18D Allelic Ladder Mix and CC5 Internal Lane Standard 500 Always include a negative control reaction to detect reagent contamination We highly recommend the use of gloves and aerosol resistant pipette tips Some reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly 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 3 B Spectral Calibration Proper spectral calibration is critical to evaluate multicolor systems with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers A matrix must be generated for each individual instrument The PowerPlex 5 Dye Matrix Standards 3100 3130 is required for spectral calibration on the ABI
59. ection 4 or 9 C We have not tested other reaction plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Vortex the PowerPlex 18D 5X Primer Pair for 15 seconds before use The reaction volume was too low or too much storage card was added This system is optimized for a final reaction volume of 25 l Decreasing the reaction volume may result in suboptimal performance Positive control does not amplify Do not include a blank punch in the positive control reaction Check to be sure that the 2800M Control DNA was diluted correctly prepare a new dilution if necessary Be sure to store the 2800M Control DNA correctly see Section 4 or 9 C Improper storage of the 2800M Control DNA Poor capillary electrophoresis injection CC5 ILS 500 peaks also affected Re inject the sample Check the syringe for leakage Check the laser power Samples were not denatured completely Heat denature samples for the recommended time then cool on crushed ice or in an ice water bath immediately prior to capillary electrophoresis Do not cool samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Poor quality formamide was used Use only Hi Di formamide when analyzing samples Instrument run ended before all CC5 ILS 500 fragments or sample fragments were detected Lengthen the run time For the Applied Biosystems 3130
60. ection 6 B or imported in Section 6 C 8 Select Analyze green arrow button to start data analysis Note By default the software displays the Analysis Requirement Summary Allelic Ladder Analysis Summary and Analysis Summary windows after quality review by the software Ensure that all requirements are met as each window appears If you do not have the Analysis Requirement Summary window activated you may need to do additional manual troubleshooting Note Sizing of alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL 6 F PowerPlex 18D Panels and Bins Text Files with GeneMapper ID Software Version 3 2 To facilitate analysis of data generated with the PowerPlex 18D System we have created panels and bins text 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 For analysis using GeneMapper ID software version 3 2 you will need the proper panels and bins text files PowerPlex_18D_Panels_vX x txt and PowerPlex_18D_Bins_vX x txt files where X x refers to the most recent version of the panels and bins text
61. ed 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 Size standard not called Starting data point was incorrect for the partial range chosen correctly in Section 6 I 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 CC5 ILS 500 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 amplitude threshold in the analysis method for the orange channel to include peaks If peaks are of 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 s
62. er ID Software Version 3 2 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 H 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 16 The type of analysis method selected must match the type of analysis method created earlier Select OK 5 Enter a detailed name such as CC5 ILS 500 advanced in the Size Standard Editor Figure 17 6 Choose Orange for the Size Standard Dye 7 Enter the sizes of the internal lane standard fragments 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases 8 Select OK 5725TA Figure 16 The Select Dye and Analysis Method window 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 TMD031 Printed in USA Revised 5 14 Page 36 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 36 6 H Importing the CC5 ILS 500 Size Standard into GeneMapper ID Software Version 3 2 The CC5_ILS_500 xml file is available for download at www promega com geneticidtools panels_bins Save the CC5_ILS_500 xml file to a known location on your computer 1 Select Tools then GeneMapper Manager 2 Select the Size Stand
63. ertain to those samples 16 Select Link Plate for Run 17 The Load Plate window will appear Select Yes 18 In the Run Information window Figure 11 assign a Run Name Select Start Run not shown 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or 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 centrifuge compatible with 96 well plates 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 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precaut
64. es on Bode Buccal DNA Collector devices one punch per 25 l amplification reaction Blood and buccal samples on nonFTA card punches e g S amp S 903 one punch per 25 l amplification reaction Pretreat nonFTA sample types with the PunchSolution Kit Cat DC9271 to lyse nonFTA samples before adding the PCR amplification mix For more information see the PunchSolution Kit Technical Manual TMD038 Failure to pretreat these samples may result in incomplete profiles Use a manual punch tool with a 1 2mm tip to manually create sample disks from a storage card Place tip near the center of the sample spot and with a twisting or pressing action cut a 1 2mm sample disk Use the plunger to eject the disk into the appropriate well of a reaction plate Automated punchers also can be used to create sample disks Refer to the user s guide for your instrument for assistance with generating 1 2mm disks technical advice and troubleshooting information Note Static may be problematic when adding a punch to a well For FTA card punches adding PCR amplification mix to the well before adding the punch may help alleviate static problems Amplification Setup 1 Thaw the PowerPlex D 5X Master Mix and PowerPlex 18D 5X Primer Pair Mix completely Note Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after
65. f 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 9650TA Figure 20 The GeneMapper ID Allele tab 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 TMD031 Revised 5 14 Page 41 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 41 6 J Creating a No Global Filter Analysis Method with GeneMapper ID Software Version 3 2 continued 10 Select the Peak Detector tab We recommend the settings shown in Figure 19 Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU and should be determined by individual laboratories Peak heights for the CC5 ILS are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower
66. file then select Import 6 Select Done to save changes and close the GeneMapper ID X Manager 6 D Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 These instructions are intended as a guide to start analyzing data in GeneMapper ID X software They are not intended as a comprehensive guide for using the GeneMapper ID X software We recommend that users contact Applied Biosystems for training on the software 1 Select Tools then GeneMapper ID X Manager 2 Select the Analysis Methods tab 3 Select New and a new analysis method dialog box will open 4 In the Analysis Method Editor window select GeneMapper ID X Security Group as the Security Group This allows access for all users of the software Other security groups may be used 5 Enter a descriptive name for the analysis method such as PowerPlex18D 20 Filter Page 29 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 29 6 D Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Version 1 2 continued 6 Select the Allele tab Figure 13 7 Select the bins text file that was imported in Section 6 A 8 Ensure that the Use marker specific stutter ratio and distance if available box is checked 9 We recommend the values shown in Figure 13 for proper filtering of stutter peaks when using the PowerPlex 18D System You may
67. files Getting Started 1 To obtain the panels and bins text files for the PowerPlex 18D System go to www promega com geneticidtools panels_bins 2 Enter your contact information and select GeneMapper ID Select Submit 3 Save the PowerPlex_18D_Panels_X x txt and PowerPlex_18D_Bins_X x txt files to a known location on your computer Importing Panels and Bins Text 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 Manager icon in the upper left navigation pane 4 Select File then Import Panels Page 35 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 35 6 F PowerPlex 18D Panels and Bins Text Files with GeneMapper ID Software Version 3 2 continued 5 Navigate to the panels text file downloaded in the Getting Started section Select the file then Import 6 In the navigation pane highlight the PowerPlex 18D panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text file downloaded in the Getting Started section above Select the file then Import 9 At the bottom of the Panel Manager window select OK The Panel Manager window will close automatically 6 G Creating a Size Standard with GeneMapp
68. formation 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 Template HIDFragmentAnalysis36_POP4 Injection Time1 5 seconds Injection Voltage1 3kV Run Time 1 500 seconds Dye Set2 G5 1Instrument sensitivities and migration times 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 2The Dye Set setting is changed in the Protocol Manager 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 TMD031 Revised 5 14 Page 25 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 25 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 continued 6 In the spectral viewer select dye set G5 and confirm that the active dye set is the file generated fo
69. imer Pair Mix 25 l 2800M Control DNA 10ng l 5 1 250 l Water Amplification Grade Post amplification Components Box 100 l PowerPlex 18D Allelic Ladder Mix 2 300 l CC5 Internal Lane Standard 500 Page 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 TMD031 Revised 5 14 Amplification Setup Thermal Cycling Instrument Setup and Sample Preparation Data Analysis Section 4 and 9 C Section 5 Section 6 Sections 4 and 9 C GeneAmp PCR System 9700 Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Section 5 B GeneMapper ID X Software Version 1 2 ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 Section 5 B Figure 1 An overview of the PowerPlex 18D System protocol GeneMapper ID Software Version 3 2 Applied Biosystems 3500 or 3500xL Genetic Analyzer Section 5 A TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 3 2 Product Components and Storage Conditions continued Product Size Cat PowerPlex 18D System 800 reactions DC1808 Not For Medical Diagnostic Use This system contains sufficient reagents for 800 reactions of 25 l each Includes Pre amplification Components Box 4 1ml PowerPlex D 5X Master Mix 4
70. ions when handling this substance Always wear gloves and safety glasses when working with formamide 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 TMD031 Revised 5 14 Page 23 Figure 11 Assigning a run name 9256TA TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 23 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 TMD031 Printed in USA Revised 5 14 Page 24 5 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 continued Sample Preparation 1 Prepare a loading cocktail by combining and mixing CC5 Internal Lane Standard 500 and Hi Di formamide as follows 1 0 l CC5 ILS 500 injections 10 0 l Hi Di formamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased 0 5 2 0 l to adjust the intensity of the size standard peaks Keep the volume of formamide at 10 0 l per well and adjust the volume added to the wells in Step 3 accordingly 2 Vortex for 10 15 seconds
71. is a registered trademark of US Dept of Health and Human Services GeneAmp is a registered trademark of Roche Molecular Systems Inc Hi Di is a trademark of Applera Corporation Macintosh is a registered trademark of Apple Computer Inc POP 4 is a registered trademark of Life Technologies Corporation Sampact is a trademark of Fitzco Vacutainer is a registered trademark of Becton Dickinson and Company Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products Promega Corporation 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 TMD031 Revised 5 14 Page 69 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 69
72. ive plate name Select the plate type HID from the drop down menu 10 Select Assign Plate Contents Figure 10 11 Assign sample names to wells 12 In the lower left portion of the screen under Assays use the Add from Library option to select the Assay created in Step 5 or one previously created Click on the Add to Plate button and close the window Note Edit the Instrument Protocol using the instructions in Step 5 A Step 2 Otherwise changes to the Instrument Protocol such as lengthening the run time made in the Assay window may not be incorporated into the instrument run 13 Under File Name Convention use the Add from Library option to select the File Name Convention created in Step 6 or one previously created Click on the Add to Plate button and close the window 14 Under Results Groups use the Add from Library option to select the Results Group created in Step 7 or one previously created Click on the Add to Plate button and close the window Figure 10 Assigning plate contents 9255TA 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 TMD031 Printed in USA Revised 5 14 Page 22 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 22 15 Highlight the sample wells then select the boxes in the Assays File Name Conventions and Results Groups that p
73. lity allelic ladders are used for analysis 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 X 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 6 D or 6 E Panels text file selected for analysis was incorrect for the STR system used Assign correct panels text 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 internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 54 Page 55 Symptoms Causes and Comments Size standard not called Starting data point was incorrect for the partial range chosen correctly in Section 6 E Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Extra peaks in 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 shor
74. ly in this section 6 In the Panel column select the panels text file that was imported in Section 6 F 7 In the Size Standard column select the size standard that was created in Section 6 G or imported in Section 6 H 8 Select Analyze green arrow button to start the data analysis Note Sizing of alleles 475 bases will not use Local Southern Method For Penta E alleles gt 24 will be labeled as OL 6 J Creating a No Global Filter Analysis Method with GeneMapper ID Software Version 3 2 These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 5 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 GeneMapper ID software Contact Applied Biosystems 5 Enter a descriptive name for the analysis method such as PowerPlex18D 6 Select the Allele tab Figure 20 7 Select the bins text file that was imported in Section 6 F TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 40 8 Ensure that the Use marker specific stutter ratio if available box is checked 9 Enter the values shown in Figure 20 for proper filtering of stutter peaks when using the PowerPlex 18D System For an explanation of the proper usage and effects o
75. nation Keep all pre amplification and post amplification 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 7 A Amplification Setup 1 Thaw the PowerPlex D 5X Master Mix and PowerPlex 18D 5X Primer Pair Mix completely Note Centrifuge tubes briefly to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 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 consume a small amount of each reagent it ensures that you have enough PCR amplification mix for all samples It also ensures that each reaction contains the same PCR amplification mix 3 Use a clean 0 2ml MicroAmp plate for reaction assembly and label appropriately Alternatively determine the number of clean 0 2ml reaction tubes required and label appropriately 4 Add the final volume of each reagent listed in Table 7 to a sterile tube Promega Corporation 2800 Woods Hollow Road Madison WI 5
76. nt Protocol navigate to the Library select Instrument Protocol then select Create Alternatively a previously created Instrument Protocol may be used Figure 3 shows the settings used at Promega for the Applied Biosystems 3500xL Genetic Analyzer for the application type dye set capillary length polymer run module and appropriate protocol information The only settings that were changed from the default settings are dye set The recommended settings are Figure 3 The Create New Instrument Protocol window 9393TA Application Type HID Capillary Length 36cm Polymer POP 4 Dye Set G5 Promega G5 spectral Run Module HID36_POP4 xl Injection Time1 24 seconds Injection Voltage 1 2kV Run Time 1 210 1 500 seconds 1Injection time may be modified 2 24 seconds to increase or decrease peak heights TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 16 Page 17 When creating an Instrument Protocol be sure to select the same dye set that was used to perform the Promega 5 dye spectral calibration We recommend using a run time of 1 210 1 500 seconds and the default injection conditions Run time and other instrument settings should be optimized and validated in your laboratory When optimizing injection conditions in your laboratory you may choose to create specific Instrument Protocols for each condition tested If a single Instrument Protocol is used follow the instructions in the
77. oaded in the Getting Started Section Select the file then Import 6 In the navigation pane highlight the PowerPlex 18D panels folder that you just imported in Step 5 7 Select File then Import Bin Set 8 Navigate to the bins text file downloaded in the Getting Started Section Select the file then Import 9 In the navigation pane highlight the PowerPlex 18D panels folder that you just imported in Step 5 10 Select File then Import Marker Stutter A warning box will appear asking if you want to overwrite current values Select Yes 11 Navigate to the stutter file downloaded in the Getting Started Section Select the file then Import 12 At the bottom of the Panel Manager window select OK This will save the panels bins and stutter text files and close the window 6 B Creating a Size Standard with GeneMapper ID X Software Version 1 2 There are two options when creating a size standard Use this protocol or the alternative protocol in Section 6 C 1 Select Tools then GeneMapper ID X Manager 2 Select the Size Standard tab 3 Select New Page 27 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 27 6 B Creating a Size Standard with GeneMapper ID X Software Version 1 2 continued 4 In the Size Standard Editor window Figure 12 select GeneMapper ID X Security Group as the Security Group This allows acces
78. or services Disclaimer of Warranties GE Healthcare Bio Sciences Corp provides no warranties to end user statutory or implied including without limitation as to product quality condition description merchantability or fitness for a particular purpose and all such warranties are hereby expressly disclaimed GE Healthcare Bio Sciences Corp hereby expressly disclaims any warranty regarding results obtained through the use of the products including without limitation any claim of inaccurate invalid or incomplete results Exclusion of Liability GE Healthcare Bio Sciences Corp and its affiliates shall have no liability to an End User including without limitation for any loss of use or profits business interruption or any consequential incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an action in contract tort strict product liability or otherwise 2011 2012 2014 Promega Corporation All Rights Reserved PowerPlex is a registered trademark of Promega Corporation DNA IQ PunchSolution and SwabSolution are trademarks of Promega Corporation ABI PRISM Applied Biosystems GeneMapper and MicroAmp are registered trademarks of Applied Biosystems Bode Buccal DNA Collector is a trademark of the Bode Technology Group Inc EasiCollect and OmniSwab are trademarks of Whatman FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GenBank
79. punches from blank paper or all color channels samples and include a reaction with one or two blank punches as a negative control Amplification of processed punches with high amounts of DNA can result in artifact peaks due to overamplification resulting in saturating signal on the CE instrument We recommend one 1 2mm punch per 25 l reaction Use of a larger punch size or a smaller reaction volume may result in overamplification and signal saturation If the signal is saturated repeat the amplification with a smaller punch a larger reaction volume or reduced cycle number Amplification of excess template for a given cycle number can result in overloading of the capillary upon electrokinetic injection The presence of excess DNA in the capillary makes it difficult to maintain the DNA in a denatured single stranded state Some single stranded DNA renatures and becomes double stranded Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis and appears as shadow peaks migrating in front of the main peaks If this occurs at a heterozygous locus it is sometimes possible to see two shadow peaks that differ in size from one another by approximately the same distance as the single stranded alleles Artifacts of STR amplification Direct amplification of gt 20ng of template can result in a higher number of artifact peaks Use the recommended punch size and number of punches Optimize the cycle number
80. r Direct Amplification Using the PowerPlex 18D System 6 A Direct Amplification of DNA from Storage Card Punches 6 B Direct Amplification of DNA from Swabs 10 5 Instrument Setup and Sample Preparation 13 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer 13 B Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 23 6 Data Analysis 26 A PowerPlex 18D Panels Bins and Stutter Text Files with GeneMapper ID X Software Version 1 2 26 B Creating a Size Standard with GeneMapper ID X Software Version 1 2 27 C Importing the CC5 ILS 500 IDX Size Standard into GeneMapper ID X Software Version 1 2 29 D Creating a Databasing or Paternity Analysis Method with GeneMapper ID X Software Ve
81. r of Reactions Final Volume Water Amplification Grade to a final volume of 25 0 l PowerPlex D 5X Master Mix 5 0 l PowerPlex 18D 5X Primer Pair Mix 5 0 l template DNA 1 0ng 2 3 4 up to 15 0 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex D 5X Master Mix and PowerPlex 18D 5X Primer Pair Mix The template DNA will be added at Step 6 2Store DNA templates in nuclease free water TE 4 buffer 10mM Tris HCl pH 8 0 0 1mM EDTA or TE 4 buffer with 20 g ml glycogen 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 3Apparent DNA concentrations can differ depending on the DNA quantification method used 24 The amount of DNA template recommended here is based on DNA concentrations determined by measuring absorbance at 260nm We strongly recommend that you perform experiments to determine the optimal DNA amount based on your DNA quantification method 4Alternatively 5ng of DNA template can be used but the
82. r the PowerPlex 5 dye chemistry It is critical to select the correct G5 spectral for the PowerPlex 5 dye chemistry If the PowerPlex 5 dye chemistry is not the active dye set locate the PowerPlex 5 dye spectral in the List of Calibrations for Dye Set G5 and select Set 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 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 40 minutes 6 Data Analysis 6 A PowerPlex 18D Panels Bins and Stutter Text Files with GeneMapper ID X Software Version 1 2 To facilitate analysis of data generated with the PowerPlex 18D System we have created panels and bins text files to allow automatic assignment of genotypes using GeneMapper ID X software We recommend that users receive training from Applied Biosystems on the GeneMapper ID X software to familiarize themselves with proper operation of th
83. ration was ineffective See Section 7 for more information on how to minimize these artifacts Table 3 Artifact Peaks Observed with the PowerPlex 18D System Loci Artifact Sizes D3S13581 Increased stutter D21S112 n 2 n 2 D5S8182 n 1 n 2 D5S8181 One or more peaks between 140 and 151 bases D7S8201 214 bases 247 bases D16S5393 n 20 D2S13383 n 20 1Artifact has been observed in direct amplifications with large amounts of template DNA 2One or two bases above and below the true allele peak 3Artifact is due to secondary structure which can occur in particularly intense or poorly denatured 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 TMD031 Revised 5 14 Page 45 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 45 7 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 7 A Amplification and Fragment Detection Symptoms Causes and Comments Faint or absent allele peaks The PowerPlex D 5X Master Mix was not vortexed well before use Vortex the 5X Master Mix for 5 10 seconds before dispensing into the PCR amplification mix Thermal cycler or plate problems Review the thermal cycling protocol in S
84. re the temperature can fluctuate Do not refreeze avoid multiple freeze thaw cycles as this may reduce activity Active protease from the SwabSolution Reagent carried over into the amplification reaction Ensure that the heat block is heating to 70 C 90 C if using a 2 2ml Square Well Deep Well Plate and samples were incubated for the full 30 minutes Incubation for shorter time periods may result in incomplete protease inactivation Do not use an incubator set at 70 C to incubate tubes or plates Heat transfer is inefficient and will result in poor performance Only use a heat block to maintain efficient heat transfer We have tested 60 minute incubation times and observed no difference in performance compared to a 30 minute incubation Faint or absent peaks for the If the positive control reaction failed to amplify check to positive control reaction make sure that the correct amount of 2800M Control DNA was added to the reaction Due to the reduced cycle numbers used with swab extracts it is necessary to increase the mass of 2800M Control DNA to obtain a profile We recommend 5ng of 2800M Control DNA per 25 l amplification reaction This mass of DNA should be reduced if the cycle number used is increased and decreased if the cycle number is increased Increase or decrease by twofold the mass of 2800M Control DNA for every one cycle decrease or increase respectively Extra peaks visible in one Swab extract was contaminated Include a
85. rmamide injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased 0 5 2 0 l to adjust the intensity of the size standard peaks Keep the volume of formamide at 10 l per well and adjust the volume added to the wells in Step 3 accordingly 2 Vortex for 10 15 seconds to mix 3 Pipet 11 l of formamide internal lane standard mix into each well 4 Add 1 l of amplified sample or 1 l of PowerPlex 18D 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 To modify the injection time or injection voltage in the run module select Instrument Protocol from the Library menu in the data collection software If peak heights are higher than desired use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program to achieve the desired signal intensity 5 Centrifuge plate briefly to remove air bubbles from the wells 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 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
86. rotocols 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 Butler J M 2005 Forensic DNA Typing 2nd ed Elsevier Academic Press London 10 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 Promega Corporation 245 69 11 Hartmann J M et al 1991 Guidelines for a quality assurance program for DNA analysis Crime Laboratory Digest 18 44 75 12 Internal Validation of STR Systems Reference Manual GE053 Promega Corporation 13 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 14 Schl tterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 15 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleoti
87. roxidase gene AATG D8S1179 TMR ET 8q24 13 NA TCTA Complex 18 vWA TMR ET 12p13 31 HUMVWFA31 human von Willebrand factor gene TCTA Complex 18 Amelogenin3 TMR ET 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 D2S1338 CXR ET 2q35 TGCC TTCC D19S433 CXR ET 19q12 AAGG Complex 1Information about chromosomal location of these loci can be found in references 19 and 20 and at www cstl nist gov biotech strbase chrom htm 2The August 1997 report 21 22 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 3Amelogenin is not an STR but displays a 109 base X specific band and a 115 base Y specific band NA not applicable Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 27
88. rsion 1 2 29 E Creating a No Global Filter Analysis Method with GeneMapper ID X Software Version 1 2 32 F PowerPlex 18D Panels and Bins Text Files with GeneMapper ID Software Version 3 2 35 G Creating a Size Standard with GeneMapper ID Software Version 3 2 36 H Importing the CC5 ILS 500 Size Standard into GeneMapper ID Software Version 3 2 37 I Creating a Databasing or Paternity Analysis Method with GeneMapper ID Software Version 3 2 38 J Creating a No Global Filter Analysis Method with GeneMapper ID Software Version 3 2 40 K Controls 43 L Results 43 Page 1 TMD031 0514 TMD031 0514 5 28 2014 12 18 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
89. rument preparation instructions in Section 5 A Step 2 and Section 5 B Step 6 Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix is applied to the samples Perform a new spectral calibration and re run the samples Instrument sensitivities can vary Optimize the injection conditions See Section 5 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 fluorescein and JOE channels Use autoclaved water change vials and wash buffer reservoir Page 47 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 47 7 A Amplification and Fragment Detection continued Symptoms Causes and Comments Extra peaks visible in one Repeat sample preparation using fresh formamide Long term or all color channels continued storage of amplified sample in formamide can result in degradation 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 Allelic ladder not running 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 Poor quality formamide Use only Hi Di formamide when analyzing samples Be sure the allelic ladder and samples are from
90. s for all users of the software Other security groups may be used 5 Enter a detailed name such as CC5_ILS_500_IDX 6 Choose Orange for the Size Standard Dye 7 Enter the sizes of the internal lane standard fragments 60 65 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Section 9 B Figure 23 8 Select OK 8257TA Figure 12 The GeneMapper ID X Size Standard Editor 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 TMD031 Printed in USA Revised 5 14 Page 28 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM 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 Printed in USA Part TMD031 Revised 5 14 6 C Importing the CC5 ILS 500 IDX Size Standard into GeneMapper ID X Software Version 1 2 The CC5_ILS_500_IDX xml file is available for download at www promega com geneticidtools panels_bins Save the CC5_ILS_500_IDX xml file to a known location on your computer 1 Select Tools then GeneMapper ID X Manager 2 Select the Size Standard tab 3 Select Import 4 Navigate to the location of the CC5_ILS_500_IDX xml file on your computer 5 Highlight the
91. stems 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 Part TMD031 Printed in USA Revised 5 14 Page 38 9651TA Figure 18 The GeneMapper ID Allele tab with settings for using a 20 peak filter TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 38 10 Select the Peak Detector tab We recommend the settings shown in Figure 19 Notes 1 Select full range or partial range for the analysis range When using a partial range choose an appropriate analysis range based on your data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The peak amplitude thresholds are the minimum peak heights at which the software will call a peak Values for peak amplitude thresholds are usually 50 150RFU and should be determined by individual laboratories Peak heights for the CC5 ILS are generally lower than those for the other dyes Therefore the threshold for the orange dye may be lower than that for the other dyes 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
92. systems 3500 or 3500xL Genetic Analyzer MicroAmp optical 96 well plate or equivalent and septa Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex 5 Dye Matrix Standards 3100 3130 Cat DG4700 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 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 TMD031 Revised 5 14 Page 13 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 13 5 A Detection of Amplified Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer continued Sample Preparation 1 Prepare a loading cocktail by combining and mixing CC5 Internal Lane Standard 500 and Hi Di formamide as follows 1 0 l CC5 ILS 500 injections 10 0 l Hi Di fo
93. t and larger peaks in ILS were not captured Not all CC5 ILS 500 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 amplitude threshold in the analysis method for the orange channel to include peaks If peaks are low quality redefine the size standard for the sample to skip these peaks Significantly raised baseline Poor spectral calibration Perform a new spectral calibration and re run the samples Incorrect G5 spectral was active Re run samples and confirm that the PowerPlex 5 dye G5 spectral is set for G5 See instructions for instrument preparation in Section 5 7 E GeneMapper ID 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 To analyze samples with GeneMapper ID software at least one allelic ladder must be defined An insufficient number of CC5 ILS 500 fragments was defined Be sure to define at least one CC5 ILS 500 fragment smaller than the smallest sample peak or allelic ladder peak and at least one CC5 ILS 500 fragment larger than the largest sample peak or allelic ladder peak R
94. 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 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 TMD031 Revised 5 14 TMD031 0514 TMD031 0514 5 28 2014 12 18 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 TMD031 Printed in USA Revised 5 14 8 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 tetrameric 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 b 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 P
95. 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 20ng of template can result in an imbalance with smaller loci showing more product than larger loci Use less template DNA or decrease number of cycles Insufficient template DNA Use the recommended amount of template DNA if available Stochastic effects can occur when amplifying low amounts of template Miscellaneous balance problems Thaw the 5X Primer Pair Mix and 5X Master Mix completely and vortex for 15 seconds before use Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after mixing Calibrate thermal cyclers and pipettes routinely PCR amplification mix prepared in Section 4 or 9 C was not mixed well Vortex the PCR amplification mix for 5 10 seconds before dispensing into the reaction plate 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 Part TMD031 Printed in USA Revised 5 14 Page 48 TMD031 051
96. tion control pipet Water Amplification Grade or TE 4 buffer instead of swab extract into a reaction well containing PCR amplification mix Note Additional negative controls can be included Assemble a reaction containing the swab extract prepared from a blank swab or assemble a reaction where the SwabSolution or PunchSolution Reagent is processed as a blank without a swab 9 Seal the plate Optional Briefly centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles 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 TMD031 Revised 5 14 Table 2 PCR Amplification Mix for Direct Amplification of DNA From Swabs PCR Amplification Mix Component1 Volume Per Reaction Number of Reactions Final Volume Water Amplification Grade 13 l PowerPlex D 5X Master Mix 5 0 l PowerPlex 18D 5X Primer Pair Mix 5 0 l swab extract 2 0 l total reaction volume 25 l 1Add Water Amplification Grade to the tube first then add PowerPlex D 5X Master Mix and PowerPlex 18D 5X Primer Pair Mix The swab extract will be added at Step 6 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 11 4 B Direct Amplification of DNA from Swabs continued Thermal Cycling Amplification and detection instrumentation may
97. to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips PunchSolution Kit Cat DC9271 for nonFTA card punches 1 2mm Harris Micro Punch or equivalent manual punch and cutting mat This section contains a protocol for direct amplification of DNA from storage card punches using the PowerPlex 18D System and GeneAmp PCR System 9700 thermal cycler Note You will need to optimize and validate the number of storage card punches per reaction in your laboratory FTA based sample types include Buccal cells collected on FTA cards with Whatman EasiCollect or Fitzco Sampact devices one or two punches per 25 l amplification reaction Buccal cells collected with sterile swabs transferred to FTA or Indicating FTA cards one or two punches per 25 l amplification reaction Liquid blood from collection or storage Vacutainer tubes or finger sticks spotted onto FTA cards one punch per 25 l amplification reaction 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 TMD031 Printed in USA Revised 5 14 Page 6 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 6 NonFTA sample types include Buccal sampl
98. to mix 3 Pipet 11 l of formamide internal lane standard mix into each well 4 Add 1 l of amplified sample or 1 l of PowerPlex 18D Allelic Ladder Mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be adjusted Use the Module Manager in the data collection software to modify the injection time or voltage in the run module see Instrument Preparation below 5 Centrifuge plate briefly to remove air bubbles from the wells 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 user s 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 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 with the following exceptions TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 24 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
99. uble stranded Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis and appears as shadow peaks migrating in front of the main peaks If this occurs at a heterozygous locus it is possible to observe the presence of two shadow peaks that differ in size by approximately the same distance as the single stranded alleles Peak height imbalance Excess DNA in the amplification reaction can result in locus to locus imbalance within a dye channel such that the peak heights at the smaller loci are greater than those at the larger loci ski slope effect Use less swab extract or reduce the cycle number Active protease carried over from swab extracts into the amplification reaction Larger loci are most susceptible to protease carryover and will drop out before smaller loci Ensure that the heat block is heating to 70 C 90 C if using 2 2ml Square Well Deep Well Plates and samples were incubated for the full 30 minutes Incubation for shorter time periods may result in incomplete protease inactivation Do not use an incubator set at 70 C to incubate tubes or plates Heat transfer is inefficient and will result in poor performance Only use a heat block to maintain efficient heat transfer Inactive SwabSolution Reagent Thaw the SwabSolution Reagent completely in a 37 C water bath and mix by gentle inversion Store the SwabSolution Reagent at 2 10 C Do not store reagents in the refriger
100. un was too short and larger peaks in ILS were not captured Not all CC5 ILS 500 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 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 TMD031 Revised 5 14 TMD031 0514 TMD031 0514 5 28 2014 12 18 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 TMD031 Printed in USA Revised 5 14 7 E GeneMapper ID Software continued Symptoms Causes and Comments 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 6 I or 6 J Panels text file file selected for analysis was incorrect for the STR system used Assign correct panels text file that corresponds to the STR system used for amplification The allelic ladder was not identifi
101. werPlex CS7 System 100 reactions DC6613 PowerPlex Y23 System 50 reactions DC2305 200 reactions DC2320 Not for Medical Diagnostic Use Additional monoplexes are available Accessory Components Product Size Cat PowerPlex 5 Dye Matrix Standards 310 50 l each dye DG4600 PowerPlex 5 Dye Matrix Standards 3100 3130 25 l each dye DG4700 2800M Control DNA 10ng l 25 l DD7101 2800M Control DNA 0 25ng l 500 l DD7251 SwabSolution Kit 100 preparations DC8271 PunchSolution Kit 100 preparations DC9271 CC5 Internal Lane Standard 500 300 l DG1521 Water Amplification Grade 6 250 l 5 1 250 l DW0991 Not for Medical Diagnostic Use 9 F Summary of Changes The following change was made to the 5 14 revision of this document Legal disclaimers were updated 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 TMD031 Revised 5 14 Page 67 TMD031 0514 TMD031 0514 5 28 2014 12 18 PM Page 67 a U S Pat No 6 242 235 Australian Pat No 761757 Canadian Pat No 2 335 153 Chinese Pat No ZL99808861 7 Hong Kong Pat No HK 1040262 Japanese Pat No 3673175 European Pat No 1088060 and other patents pending b U S Pat Nos 5 843 660 6 479 235 6 221 598 and 7 008 771 Australian Pat No 724531 Canadian Pat

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