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StemElite™ ID System

1. 7818TA Figure 6 Representative data for the StemElite ID System Genomic DNA from 9947A cells 2 0ng combined with 20pg of mouse genomic DNA prior to amplification was amplified using the StemElite ID System Amplification products were mixed with Internal Lane Standard 600 and analyzed with an Applied Biosystems 3130x Genetic Analyzer using a 1 5 kV 5 second injection The results were analyzed using GeneMapper ID software version 3 2 and the appropriate panel and bin files Artifacts and Stutter Stutter bands are a common amplification artifact associated with STR analysis Stutter products are often observed one repeat unit below the true allele peak and occasionally two repeat units smaller or one repeat unit larger than the true allele peak Frequently alleles with a greater number of repeat units will exhibit a higher percent stutter The pattern and intensity of stutter can differ slightly between primer sets for the same loci The degree of stutter for the STR loci used in StemElite ID System was determined and published as part of the PowerPlex 16 System validation 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM307 Printed in USA Page 24 10 08 Promega fy ee c Mo E M LM UM eee aaa aaa 44o 2L LA LN MI UM 2 ZG c 8252 2128 2 a pue 322 d 38 2 ai 38
2. Factory Defaults 7264TA Figure 5 GeneMapper ID and GeneMapper Analysis Method Peak Detector tab 10 D Creating a Size Standard 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 5 The type of analysis method selected must match the type of analysis method created earlier Select OK 5 Enter a detailed name such as ILS 600 advanced in the Size Standard Editor Choose red as the color for the size standard dye 6 Enter the sizes of the internal lane standard fragments 7 Select OK Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM307 10 08 Page 21 10 E Processing Data 1 7 Import sample files into a new project as described in the Applied Biosystems GeneMapper ID software human identification analysis tutorial or GeneMapper Software Version 4 0 Microsatellite Analysis Getting Started Guide In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control Every folder in the project must contain at least one ladder that is designated as such for proper genotyping In the Analysis Method column select the analysis method created in Section 10 C In the Panel column
3. Amplification Grade to a final volume of 25 0p1 template DNA 2ng up to 17 5pl The template DNA will be added at Step 7 Amplification of gt 2ng of DNA template results in an imbalance in peak heights from locus to locus in the resulting data The smaller loci show greater amplification yield than the larger loci Reducing the number of cycles in the amplification program by 2 4 cycles i e 10 20 or 10 18 cycling can improve locus to locus balance 5 Vortex the PCR master mix for 5 10 seconds 6 Pipet PCR master mix into each reaction tube 7 Pipet the template DNA 2ng for each sample into the respective tube containing PCR master mix 8 For the positive amplification control dilute the 9947A DNA to 2ng in the desired template DNA volume Pipet 2ng of the diluted DNA into a reaction tube containing the PCR master mix 9 Optional If including a positive amplification control for mouse DNA dilute the Mouse Genomic DNA Cat G3091 must be purchased separately to 100pg in the desired template DNA volume Pipet 100pg of the diluted DNA into a reaction tube containing the PCR master mix 10 For the negative amplification control pipet Water Amplification Grade instead of template DNA into a reaction tube containing the PCR master mix Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part
4. Thaw the StemElite ID 5X Enzyme Mix and StemElite ID 10X Primer Pair Mix Note Vortex reagents for 15 seconds before each use Do not centrifuge the 10X Primer Pair Mix as this may cause the primers to be concentrated at the bottom of the tube 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 TM307 Printed in USA Page 8 10 08 2 Determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for pipetting error While this approach does waste a small amount of each reagent it ensures that you will have enough PCR master mix for all samples It also ensures that each reaction contains the same master mix 3 Place one clean 0 2ml reaction tube for each reaction into a rack and label appropriately Alternatively use a MicroAmp plate or 0 2ml MicroAmp 8 strip reaction tubes and label appropriately 4 Add the final volume of each reagent listed in Table 1 into a sterile 1 5ml amber colored tube Mix gently A worksheet to calculate reagent volumes for a multiple reaction StemElite D master mix can be found in Section 12 Table 1 PCR Master Mix for the StemElite ID System PCR Master Mix Component Volume Per Reaction StemElite D 5X Enzyme Mix 5 0pl StemElite D 10X Primer Pair Mix 2 9 Water
5. select StemElite ID Panels 1 0 This is the panel set that was imported in Section 10 B In the Size Standard column select the size standard that was created in Section 10 D If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column Select Analyze green arrow button to start the data analysis 10 F Obtaining a Genotype Sample genotypes have been generated during data processing Section 10 E 1 Select View then Genotypes Genotypes for all samples in the project will be shown Alternatively highlight a sample or a subset of samples in the project select Analysis then select Display Plots Within the Sample Plot window select Genotype Genotypes for the selected samples will be shown If you are using GeneMapper software and the sample alleles are being called as OL off ladder the bins may not be appropriate for your instrument GeneMapper software does not offset the bins to match the allelic ladder like the GeneMapper ID software does Visit the Promega web site at www promega com stemeliteidapps to generate custom bins specific for your 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 Part TM307 Page 22 Printed in USA 10 08 10 G Controls 1 Exami
6. 2 0 888885 8 8 822828888 7819TA Figure 7 The StemElite ID Allelic Ladder The StemElite ID Allelic Ladder was analyzed with an Applied Biosystems 3130xI Genetic Analyzer using a 3kV 5 second injection The sample file was analyzed with the GeneMapper ID software version 3 2 and the StemElite ID panel and bin files In addition to stutter peaks other artifact peaks can be observed at some StemElite D System loci Low level products can be seen in the n 2 and n 2 positions two bases below and above the true allele peak respectively with some loci such as D21S11 Samples may show low level artifacts in the noncalling regions between the D75820 and D135317 allele ranges and between the Mouse and THO01 allele ranges Occasionally an off ladder artifact can be observed in the 270 271bp position in the JOE dye channel One or more extra peaks that are not directly related to amplification may be observed at positions 8 26 bases smaller than TPOX alleles and 6 21 bases smaller than vWA alleles These extra peaks occur when the amplified peaks are particularly intense high signal level or template amount the formamide polymer or capillary was of poor quality or denaturation was ineffective One or more extra peaks that are not directly related to amplification may be observed at 73bp in the fluorescein channel and at 72 77bp in the JOE channel See Section 11 Troubleshooting for more information on how to mi
7. 2p23 pter HUMTPOX human AATG thyroid peroxidase gene vWA TMR 12pl12 pter HUMVWEFA31 human von TCTA Willebrand factor gene Complex 16 Amelogeni TMR Xp221 223 HUMAMEL human Y NA and Y chromosomal gene for Amelogenin like protein CSF1PO JOE 5q33 3 34 HUMCSF1PO human AGAT c fms proto oncogene for CSF 1 receptor gene D165539 JOE 16q24 qter NA GATA D75820 JOE 7q11 21 22 NA GATA D138317 JOE 13q22 q31 NA TATC D55818 JOE 5023 3 32 NA AGAT The August 1997 report 17 18 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 Mouse is not an STR but displays a mouse specific band Amelogenin is not an STR but displays a 106 base X specific band and a 112 base Y specific band TMR carboxy tetramethylrhodamine FL fluorescein JOE 6 carboxy 47 5 dichloro 27 7 dimethoxy fluorescein 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 608 277 2516 www promega com Printed in USA Part TM307 10 08 Page 33 Table 4 The StemElite ID System Allelic Ladder Information STR Locus Label D
8. Business Development Department Stratagene California 11011 North Torrey Pines Road La Jolla CA 92037 2008 Promega Corporation All Rights Reserved Apo ONE Caspase Glo CellTiter Glo MagneSil Maxwell PowerPlex and Wizard are registered trademarks of Promega Corporation Cell ID CytoTox Fluor CytoTox Glo DNA IQ Slicprep and StemElite are trademarks of Promega Corporation ABI PRISM GeneMapper GeneScan and MicroAmp are registered trademarks of Applera Corporation ART is a registered trademark of Molecular Bio Products Inc GenBank is a registered trademark of U S Dept of Health and Human Services GeneAmp is a registered trademark of Roche Molecular Systems Inc Hi Di and POP 4 are trademarks of Applera Corporation Quant iT is a trademark of Molecular Probes Inc PicoGreen is a registered trademark of Molecular Probes Inc Windows is a registered trademark of Microsoft Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products Promega Corporation 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 TM
9. System 7 Protocols for DNA Amplification Using the StemElite ID System Materials to Be Supplied by the User e GeneAmp PCR System 9600 or 9700 thermal cyclers Applied Biosystems e 0 2ml thin walled microcentrifuge tubes MicroAmp optical 96 well reaction plate or 0 2ml MicroAmp 8 strip reaction tubes Applied Biosystems e 5mlamber colored microcentrifuge tubes Fisher Cat 05 402 26 e aerosol resistant pipette tips The StemElite ID System has been optimized to amplify 2ng of template DNA in a 25yl reaction using the protocols detailed below Preferential amplification of smaller loci can occur Expect to see high peak heights for the smaller loci and relatively lower peak heights for the larger loci if more than the recommended amount of template is used Reduce the amount of template DNA or number of PCR cycles to correct this The StemElite ID System is optimized for the GeneAmp PCR system 9700 thermal cycler An amplification protocol for the GeneAmp PCR system 9600 thermal cycler is also provided 7 4 Amplification Setup Keep all pre amplification and postamplification reagents in separate rooms Prepare amplification reactions in a room dedicated to reaction setup Use equipment and supplies dedicated to amplification setup Cross contamination with another template or previously amplified DNA can lead to extra peaks in the sample Use aerosol resistant pipette tips and change gloves regularly 1
10. a different run than the samples was used Reanalyze the 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 reanalyze as described in Section 10 Panel file selected for analysis was incorrect Assign correct panel file that corresponds to the StemElite ID System The allelic ladder was not identified as an allelic ladder in the sample type column when using GeneMapper ID software The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample When using the GeneMapper software the bins may need to be adjusted for the unique migration characteristics of your instrument Use the custom bin generator 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 TM307 Printed in USA Page 30 10 08 Symptoms Causes and Comments Size standard not called Starting data point was incorrect for the partial range correctly Figure 8 chosen in Section 10 C Adjust the starting data point in the analysis method Alternatively use a full range for the analysis Run was too short and larger peaks in ILS were not detected Not all of the ILS 600 peaks
11. applied when run on analysis of samples and the following error message appears when data are displayed Some selected sample s do not contain analysis data Those sample s will not be shown Error message after attempting to import panel and bin files Unable to save panel data java SQLEException ORA 00001 unique constraint IFA CKP_NNN violated 2800 Woods the ABI PRISM 310 genetic analyzer no data will be displayed Apply a matrix file during analysis in the GeneMapper ID software and reanalyze There was a conflict between different sets of panel and bin files Delete all panel and bin sets and reimport files in a different order Hollow Road Madison WI 53711 5399 USA Promega Corporation Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Partt TM307 Printed in USA Page 32 10 08 12 Appendix 12 A Additional STR Locus Information Additional information about the human STR loci amplified by the StemElite ID System can be found in Table 3 StemElite ID System allelic ladder information can be found in Table 4 Table 3 The StemElite ID System Locus Specific Information Chromosomal GenBank Locus and Repeat Sequence STR Locus Label Location Locus Definition 53 D21S11 FL 21gl1 21g21 HUMD21LOC TCTA Complex 16 THO1 FL pibo HUMTH01 human AATG 16 tyrosine hydroxylase gene Mouse FL Mouse Mouse locus near NA chromosome 4 D4Mit113 TPOX TMR
12. d dd f Size bp 100 200 300 400 A Amelogenin z M Mouse Figure 1 Allele ranges for the StemElite ID System STR fragments amplified by the StemElite ID System are labeled with different dyes and are separated by capillary electrophoresis based on size A size standard is included in each sample to determine the size of the StemElite ID amplified fragments Fluorescein labeled loci are shown in dark gray JOE labeled loci are shown in gray and TMR labeled loci are shown in white The CXR labeled Internal Lane Standard 600 fragments are represented by black bars 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 TM307 10 08 Page 3 Q Pro ega 1 Description continued the size of each amplified product The allelic ladder consists of all major alleles at a particular locus and is used as a standard to positively identify each allele It is included in each analysis run to control for run to run variation StemElite ID allelic ladder information indicating the size range and repeat numbers for each allele can be found in Section 12 A The control 9947A DNA has a known genotype and can be used to verify genotyping accuracy Figure 2 outlines the StemElite ID System protocols in this manual Briefly DNA is isolated from cells quantitated and added to a master mix containing the St
13. data and status windows Each sample will take approximately 40 minutes for syringe pumping sample injection and sample electrophoresis Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM307 Printed in USA Page 16 10 08 10 Data Analysis To interpret data generated using the StemElite ID System you will need GeneMapper or GeneMapper ID software To facilitate analysis of data generated with the StemElite ID System we have created panel and bin files to allow automatic assignment of genotypes using GeneMapper ID software version 3 2 and GeneMapper software version 4 0 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 the proper operation of the software For GeneMapper ID software version 3 1 users we recommend upgrading to version 3 2 10 A Downloading Panel and Bin Files 1 Download the panel and bin files for use with GeneMapper ID and GeneMapper software from the Promega web site www promega com stemeliteid 2 Enter your contact information and select the appropriate analysis software Select Submit 3 Select the StemElite ID Panels and Bins Set link and save the zipped file to your computer 4 Open the files using the Windows
14. 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 Rerun the samples using a longer run time Peaks in size standard If peaks are below threshold reinject the sample missing ee If peaks are low quality redefine the size standard for the sample to skip these peaks Error message The size standard and analysis method were not in the Either panel size same mode Classic vs Basic or Advanced Be sure standard or analysis both files are set to the same mode either Classic or Basic method is invalid or Advanced mode E no export pplext6 vi adv PowerPlex 16 v1 4 ILS600 adv ST0A thc ct ce em 03 Run Folder 4 ABISTO uc MES gt gt SSCS 7 7 j a Size 11 no File Eck Wiew Took AHO i Hz e asso Standard MEE E a Quality is Sizing Quality 0 0 pO he fo JEET 7 o po JEEN 18 po r ABIS1O Ea EET AEISTO RESO grem 19 no 20 no PX no 22 no ARNO 24 no ss JEEN 26 no ABIST0 27 no rE ET 26 no ARNO 2 ho AEISTO 39 no OK Cancel Apply i ABIST0 D gt gt gt gt gt a mmmmmmmm mnmOHEHGE 5686TA it no expo pplext6 vT 4adv Powerex T5 v CSET ad nz DE TRR CXR Run Folder a AB6310 Figure 8 An example showing improper assignment of size standard fragments
15. in the GeneMapper ID software Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM307 10 08 Page 31 11 B GeneMapper and GeneMapper ID Analysis Software continued Symptoms Causes and Comments No alleles called but no error message appears Panel or size standard was not selected for sample Select the appropriate options and reanalyze Size standard was not correctly defined or size peaks were missing Check the size standard Error message Both the Bin Set used in the Analysis Method and the Panel must belong to the same Chemistry Kit The wrong bin set or a deleted bin set was chosen in the analysis method Allele tab Be sure to choose the appropriate bin set as shown in Figure 3 or 4 Significantly raised baseline Poor spectral calibration for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xl genetic analyzers Perform a new spectral calibration and rerun the samples Poor matrix for the ABI PRISM 310 genetic analyzer Rerun and optimize the matrix Use of Classic mode analysis method can result in baselines with more noise than those analyzed using the Basic or Advanced mode analysis method Advanced mode analysis methods and size standards are recommended Red bar appears during If none of the samples had matrices
16. in or preparation for legal proceedings as well as the compilation and indexing of the results of such analysis and also analysis for parentage determination the Forensic and Genetic Identity Applications Field The Forensic and Genetic Identity Applications Field specifically excludes tissue typing related to transplantation or other medical procedures Further licensing information may be obtained by contacting the USB Corporation 26111 Miles Road Cleveland Ohio 44128 This product is sold under licensing arrangements with Stratagene The purchase price of this product includes limited nontransferable rights under U S Pat Nos 5 449 603 5 605 824 5 646 019 and 5 773 257 owned by Stratagene to use only this amount of the product to practice the claims in said patent solely for activities of end users within the fields of life science research and forensic analysis of genetic material relating to or obtained as the result of criminal investigations or disaster sites conducted either by or for a governmental entity or for use in or preparation for legal proceedings as well as the compilation and indexing of the results of such analysis and also analysis for parentage determination the Forensic and Genetic Identity Applications Field The Forensic and Genetic Identity Applications Field specifically excludes tissue typing related to transplantation or other medical procedures Further licensing information may be obtained by contacting the
17. 071 Water Amplification Grade 6 250pl 5 x 1 250p1 DW0991 9947A DNA 250ng 10ng pl DD1001 Mouse Genomic DNA 100ug G3091 Not for Medical Diagnostic Use Por Laboratory Use 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 TM307 Printed in USA Page 38 10 08 Promega Cell Viability and Cytotoxicity Assays Product Size Cat CytoTox Glo Cytotoxicity Assay 10ml G9280 MultiTox Fluor Multiplex Cytotoxicity Assay 10ml G9200 CytoTox Fluor Cytotoxicity Assay 10ml G9260 CellTiter Glo Luminescent Cell Viability Assay 10ml G7570 For Laboratory Use Additional Sizes Available Apoptosis Assays Product Size Cat Apo ONE Homogeneous Caspase 3 7 Assay fluorescent 10ml G7790 Caspase Glo 3 7 Assay 10ml G8091 Caspase Glo 8 Assay 10ml G8201 Caspase Glo 9 Assay 10ml G8211 For Laboratory Use Additional Sizes Available ART Aerosol Resistant Tips Product Volume Size tips pack Cat ART 10 Ultramicro Pipet Tip 0 5 10ul 960 DY1051 ART 20E Ultramicro Pipet Tip 0 5 10ul 960 DY1061 ART 20P Pipet Tip 20yl 960 DY1071 ART GEL Gel Loading Pipet Tip 100yl 960 DY1081 ART 100 Pipet Tip 100gl 960 DY1101 ART 100E Pipet Tip 100pl 960 DY1111 ART 200 Pipet Tip 200p1 960 DY1121 ART 1000E Pipet Tip 1 0001 800 DY1131 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Fre
18. 21S11 FL TH01 FL Mouse FL TPOX TMR vWA TMR Amelogenin TMR CSF1PO JOE D16S539 JOE D75820 JOE 10135317 JOE D55818 JOE Size Range of Allelic Ladder Components bases 203 259 156 195 90 91 262 290 123 171 106 112 321 357 264 304 215 247 176 208 119 155 Repeat Numbers of Allelic Ladder Components 24 24 2 25 25 2 26 28 28 2 29 29 2 90 90 2 01 912 902 92 2 33 33 2 34 34 2 35 35 2 36 38 4 9 9 3 10 11 13 3 Mouse 6 13 10 22 XY 6 15 5 8 15 6 14 7 15 7 16 1The length of each allele in the allelic ladder has been confirmed by sequence analysis When using an internal lane standard such as the Internal Lane Standard 600 the calculated sizes of allelic ladder components may differ from those listed This occurs because different sequences in allelic ladder and ILS components may cause differences in migration The dye label also affects migration of alleles 12 B The Internal Lane Standard 600 The Internal Lane Standard ILS 600 contains 22 DNA fragments of 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases in length Figure 9 Each fragment is labeled with carboxy X rhodamine CXR and may be detected separately as a fourth color in the presence of StemElite TD amplified material The ILS 600 is designed for use in each CE injection to increase precision in analyses when using the StemElite D System Protoco
19. 274 4330 Fax 608 277 2516 www promega com Part TM307 Printed in USA Page 4 10 08 Promega 2 Product Components and Storage Conditions Product Size Cat StemElite ID System 50 reactions G9530 G9530 contains sufficient reagents for 50 reactions of 25pl each Includes Pre amplification Components Box Blue Label 250 StemElite ID 5X Enzyme Mix 125g StemElite ID 10X Primer Pair Mix 1 25ml Water Amplification Grade 25ul 9947A DNA 10ng tl Postamplification Components Box Beige Label 25ul StemElite ID Allelic Ladder 150p Internal Lane Standard ILS 600 Storage Conditions Store all components at 20 C in a nonfrost free freezer The StemElite ID 10X Primer Pair Mix StemElite ID Allelic Ladder and Internal Lane Standard 600 are light sensitive and must be stored protected from light We strongly recommend that pre amplification and postamplification reagents be stored and used separately with different pipettes tube racks etc See the expiration date on the product label 3 Instrumentation Requirements Software and Accessory Products This manual contains separate protocols for use of the StemElite ID System with GeneAmp PCR system 9700 and 9600 thermal cyclers in addition to protocols to separate amplified products and detect separated material using the capillary electrophoresis instruments ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x Gen
20. 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 TM307 10 08 Page 15 9 B Capillary Electrophoresis Instrument Preparation continued 4 Select the GS STR POP4 1ml A Module using the pull down menu Change the injection time to 3 seconds and the run time to 30 minutes Keep the settings for the remaining parameters as shown below Inj Secs 3 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time 30 You may need to optimize the injection time for individual instruments Injection times of 2 5 seconds are suggested for reactions that contain 2ng of template DNA Note Migration of fragments can vary slightly over the course of a long ABI PRISM 310 Genetic Analyzer run This may be due to changes in temperature or changes in the column When analyzing many samples injections of the allelic ladder at different times throughout the run can aid in accurately genotyping the samples 5 Select the appropriate matrix file 6 To analyze the data automatically select the auto analyze checkbox and the appropriate analysis parameters and size standard Refer to the ABI PRISM 310 Genetic Analyzer user s manual for specific information on these options 7 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system 8 Monitor the electrophoresis by observing the raw
21. 307 Printed in USA Page 40 10 08
22. Bar 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 18 Gill P et al 1997 Considerations from the European DNA Profiling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 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 TM307 10 08 Page 37 12 F Related Products Sample Preparation Systems Product Size Cat Wizard SV Genomic DNA Purification System 50 preps A2360 250 preps A2361 Wizard Genomic DNA Purification Kit 100 isolations x 300gl A1120 500 isolations x 300gl A1125 100 isolations x 10ml A1620 MagneSil Genomic Fixed Tissue System 100 samples MD1490 DNA IQ System 100 reactions DC6701 400 reactions DC6700 Slicprep 96 Device 10 pack V1391 For Laboratory Use Maxwell Automated Nucleic Acid Purification Product Size Cat Maxwell 16 Cell LEV DNA Purification Kit 48 preps AS1140 For more information about other Maxwell Nucleic Acid Purification Kits visit www promega com maxwell16 For Laboratory Use Accessory Components Product Size Cat PowerPlex Matrix Standards 310 0p each dye DG4640 PowerPlex Matrix Standards 3100 3130 25ul each dye DG4650 Internal Lane Standard 600 150ud DG1
23. TM307 10 08 Page 9 7 B Amplification Thermal Cycling This manual contains protocols for use of the StemElite ID System with the GeneAmp PCR system 9600 and 9700 thermal cyclers We have not tested other reaction tubes plates or thermal cyclers For information about other thermal cyclers please contact Promega Technical Services by e mail techserv promega com Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection time or loading volume for each laboratory instrument Testing at Promega Corporation shows that 10 22 cycles work well for 2ng of purified DNA templates For higher amounts of input DNA or to decrease sensitivity fewer cycles such as 10 16 10 18 or 10 20 should be evaluated In house validation should be performed 1 Place the tubes or MicroAmp plate in the thermal cycler 2 Select and run a recommended protocol The preferred protocols for use with the GeneAmp PCR system 9700 and 9600 thermal cyclers are provided below 3 After completion of the thermal cycling protocol store the samples at 20 C in a light protected box Note Storage of amplified samples at 4 C or higher may produce degradation products Protocol for the GeneAmp PCR Protocol for the GeneAmp PCR System 9700 Thermal Cycler System 9600 Thermal Cycler 96 C for 2 minutes then 96 C for 2 minutes then 94 C for 30 seconds then ramp 60 seconds to 60 C h
24. Technical Manual StemElite ID System INSTRUCTIONS FOR USE OF PRODUCT G9530 PRINTED IN USA 10 08 Part TM307 AF9TM307 1008TM307 All technical literature is available on the Internet at www promega com tbs Please visit the web site to verify that you are using the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail techserv promega com IM D O m 2 2 Product Components and Storage Conditions 5 3 Instrumentation Requirements Software and Accessory Products sss B 4 Matrix Standardization or Spectral Calibration 6 DEOETEERH ITOI o A A MNONOMU NEU 6 6 DNA Sample Preparation teen Neue edis 7 As DNA POIN ONON iasg siaaa Neasa AeA iiie 7 B IN FiO UD TA OM wc secs ctorsconacsensiooenaansnaae nentcaaws bat cana due ree ii eniai aor 7 7 Protocols for DNA Amplification Using the StemElite ID System 8 7 JP PAIN CAN a ede tet deus MEME Ie 8 b sip Cathy Terie Cy Clg a aiies 10 8 Detection of Amplified Fragments Using the Applied Biosystems 3130 or 3130x Genetic Analyzer and ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Versions 2 0 and 3 0 11 A Capillary Electrophoresis Sample Preparation 11 B Capillary Electrophor
25. Verify cell line identity with DNA profiling ATCC Connection Newsletter of The American Type Culture Collection 21 1 2 8 Krenke B et al 2002 Validation of a 16 locus fluorescent multiplex system J Forensic Sci 47 773 85 9 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 10 Schlotterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 11 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Tag DNA polymerase Genome Res 5 312 7 12 Magnuson V L et al 1996 Substrate nucleotide determined non templated addition of adenine by Tag DNA polymerase Implications for PCR based genotyping BioTechniques 21 700 9 13 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 14 Moller A Meyer E and Brinkmann B 1994 Different types of structural variation in STRs HumFES FPS HumVWA and HumD 1911 Int J Leg Med 106 319 23 15 Brinkmann B Moller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 16 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 17
26. WinZip program and save the unzipped files to a known location on your computer 10 B Importing Panel and Bin Files for GeneMapper ID and GeneMapper Software These instructions loosely follow the Applied Biosystems GeneMapper ID software tutorial pages 1 4 and the GeneMapper Software Version 4 0 Microsatellite Analysis Getting Started Guide 1 Open the GeneMapper ID software version 3 2 or GeneMapper software version 4 0 Select Tools then Panel Manager Highlight the Panel Manager icon in the upper left tile navigation pane Select File then Import Panels pn we O Y Navigate to the panel and bin files saved in Section 10 A Select StemElite ID Panels 1 0 txt Select Import 6 In the navigation pane highlight the StemElite ID Panels 1 0 txt folder that you just imported 7 Select File then Import Bin Set 8 Navigate to the panel and bin files Select StemElite ID Bins 1 0 txt then Import 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 Partt TM307 10 08 Page 17 10 B Importing Panel and Bin Files for GeneMapper ID and GeneMapper Software continued 9 At the bottom of the Panel Manager window select Apply then OK The panel manager window will close automatically Note GeneMapper ID software was created specifically for foren
27. ain 0 25pl of ILS 600 and 9 75ul of formamide 2 Vortex for 10 15 seconds 3 Pipet 10pl of formamide internal lane standard mix into each well Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM307 10 08 Page 11 8 A Capillary Electrophoresis Sample Preparation continued 4 Add 1pl of amplified sample or 1pl of StemElite ID Allelic Ladder Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module Alternatively use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 5 Centrifuge the plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Note Denatured sample plates should be run within 24 hours of setup Long term storage of amplified sample in formamide can result in DNA degradation Sample preparation can be repeated with fresh formamide by repeating Steps 1 6 8 B Capillary Electrophore
28. alysis tab and select GeneMapper Generic in the Analysis type drop down list 5 Place samples in the instrument and close the instrument doors 6 In the spectral viewer confirm that dye set F is active and set the correct active calibration for dye set F 7 In therun scheduler locate the plate record that you just created in Steps 3 and 4 and click once on the name to highlight it Once the plate record is highlighted click the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples 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 8 Click on the green Run Instrument arrow on the toolbar to start the sample run 9 Monitor electrophoresis by observing the run view array or capillaries viewer window in the data collection software Each injection will take approximately 45 minutes Note If peaks are low or absent the sample can be reinjected with increased injection time and or voltage If the ILS 600 is also affected check the laser power 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 TM307 10 08 Page 13 9 Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User e 95 C dry
29. amide is critical Use Hi Di formamide with a conductivity less than 100p5 cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100pS cm may contain ions that compete with DNA during injection This results in lower peak heights in the resulting data and reduced sensitivity A longer injection time may not increase the signal Caution Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 8 A Capillary Electrophoresis Sample Preparation 1 Prepare a loading cocktail by combining and mixing the internal lane standard and Hi Di formamide as follows 0 5p11 ILS 600 x injections 9 5pl Hi Di formamide x injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks in the resulting data The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If the peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0pl of ILS 600 and 9 0pl of Hi Di formamide If the peak heights are too high we recommend altering the loading cocktail to cont
30. ampler tray 48 or 96 tube 7 Place the autosampler tray in the instrument and close the instrument doors Note Denatured sample plates should be run within 24 hours of setup n Long term storage of amplified sample in formamide can result in DNA degradation Sample preparation can be repeated with fresh formamide 9 B Capillary Electrophoresis Instrument Preparation Refer to the instrument user s manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe Follow the manufacturer s recommendations for polymer storage and shelf life Polymer stored at room temperature for more than 1 week can result in broad or split peaks or extra peaks visible in one or all of the color channels Maintain the instrumentation on a daily or weekly basis as recommended by the manufacturer for optimal results and fewer instrument related artifacts Contaminants in the water used with the instrument or to dilute the 10X Genetic Analyzer buffer may generate peaks in the blue and green dye colors Use autoclaved water 1 Open the ABI PRISM 310 data collection software 2 Prepare a GeneScan sample sheet as described in the ABI PRISM 310 Genetic Analyzer user s manual Enter the appropriate sample information in the sample info column 3 Create a new GeneScan injection list Select the appropriate sample sheet by using the pull down menu Promega Corporation
31. amplification and three color detection of ten human loci nine STR loci Amelogenin for gender identification and one mouse locus including D21S11 TH01 TPOX vWA Amelogenin CSF1PO D165539 D75820 D135317 D55818 and the mouse locus These loci collectively provide a genetic profile with a random match probability of 1 in 2 92 x 10 while simultaneously providing detection of a 1 fraction of mouse contaminant in a human cell line One primer for each of the D21511 TH01 and mouse loci is labeled with fluorescein FL one primer for each of the TPOX vWA and Amelogenin loci is labeled with carboxy tetramethylrhodamine TMR and one primer for each of the CSFIPO D165539 D75820 D135317 and D55818 loci is labeled with 6 carboxy 4 5 dichloro 2 7 dimethoxy fluorescein JOE The eleven loci are amplified simultaneously in a single tube and analyzed by capillary electrophoresis in a single injection Figure 1 shows the allele ranges for each locus in the StemElite ID System In addition the StemElite ID System contains various controls to provide increased confidence in the genotypes obtained An internal lane standard ILS and allelic ladder are provided for standardization and a control cell line DNA 9947 is supplied as a positive control The ILS is added to every sample after amplification and used within each capillary electrophoresis run to determine Fluorescein DAQQA l JOE TMR vwa on
32. artial range for the analysis range When using a partial range choose an appropriate analysis range based on the data Choose a start point after the primer peak and just before the first defined internal lane standard peak to help ensure proper sizing of the internal lane standard 2 The StemElite ID System contains only tetranucleotide markers Settings for other repeat types can be ignored 11 Select the Peak Quality tab You may change the settings for peak quality Note For Steps 10 and 11 see the GeneMapper ID user s manual for more information 12 Select the Quality Flags tab You may also change these settings 13 Select DK to save your settings 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 TM307 Printed in USA Page 20 10 08 Analysis Method Editor HID Peak Detection Algorithm Advanced wv Ranges M Peak Detection Analysis Sizing Peak Amplitude Thresholds Start Size Stop Size r Smoothing and Baselining Min Peak Half Width Smoothing O None Light Polynomial Degree Heavy Peak Window Size Baseline Window 8 pts Slope Cnreriolg Feak Start r Size Calling Method O 2nd Order Least Squares O 3rd Order Least Squares Q Cubic Spline Interpolation 9 Local Southern Method Global Southern Method Peak End
33. ctions to fail Use the recommended amount of template DNA Stochastic effects which can cause imbalance can occur when amplifying low amounts of template Amplifying high amounts of template can result in less amplification of larger SIR loci Absorbance readings at 260nm can be used to estimate DNA concentration where 1AU 50g of double stranded DNA ml The Quant iI PicoGreen dsDNA quantitation assay Invitrogen can also be used Use 2ng of DNA in each StemElite ID System reaction Note As little as 20pg of mouse DNA is detectable in the StemElite ID System 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 TM307 10 08 Page 7 6 B DNA Quantitation continued If you are not using one of these kits for DNA purification we recommend measuring absorbance of the DNA sample at 260nm and 280nm to confirm that the DNA is sufficiently free of impurities High quality DNA has a typical Ax 9 Ago ratio of 1 8 The presence of impurities in the DNA sample can cause amplification failure Note that DNA concentration can be overestimated by spectrophotometry if the Azgo Avgo ratio is low Cross contamination with another template or previously amplified DNA can lead to extra peaks in the sample Use aerosol resistant pipette tips and change gloves regularly when working with the StemElite ID
34. decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If the peak heights are too low we recommend altering the ILS formamide mix to 1 5pl of ILS 600 and 24pl of Hi Di formamide If the peak heights are too high we recommend altering the loading cocktail to contain 0 5pl of ILS 600 and 24 5pl of Hi Di formamide 2 Vortex for 10 15 seconds 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 TM307 Printed in USA Page 14 10 08 O 3 Combine 25yl of the prepared loading cocktail and 1p of amplified sample or 1pl of StemElite ID Allelic Ladder Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased Alternatively use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 4 Centrifuge the sample tubes briefly to remove air bubbles from the wells if necessary 5 Denature the samples and ladder by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath for 3 minutes Note Improper denaturation can result in extra peaks 6 Assemble the tubes in the appropriate autos
35. e is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the DNA volume should not exceed 20 of the total reaction volume Store and dilute DNA in TE buffer 10mM Tris HCI pH 8 0 0 1mM EDTA or nuclease free water Thermal cycler plate or tube problems Review the thermal cycling protocols in Section 7 B We have not tested other reaction tubes plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Vortex the StemElite ID 10X Primer Pair Mix for 15 seconds before use Do not centrifuge the 10X Primer Pair after mixing Poor CE injection ILS 600 peaks also affected Reinject the sample Check the system for leakage Check the laser power Poor quality formamide was used Use only Hi Di formamide when running samples If DNA template consists of gt 50 mouse DNA content human DNA peaks may be reduced If a genotype is still required from this sample consider the use of a cell line STR system without a mouse marker such as the Cell ID System Cat G9500 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 TM307 10 08 Page 27 O 11 A Amplification and Fragment Detection continued Symptoms Causes and Comments Extra peaks visible in one Contam
36. e in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM307 10 08 Page 39 JU S Pat Nos 5 843 660 and 6 221 598 Australian Pat No 724531 Canadian Pat No 2 118 048 Korean Pat No 290332 Singapore Pat No 57050 Japanese Pat No 3602142 and other patents pending 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 Forderung der Wissenschaften e V Germany The development and use of STR loci are covered by US Pat No 5 364 759 Australian Pat No 670231 and other pending patents assigned to Baylor College of Medicine Houston Texas Patents for the foundational PCR process European Pat Nos 201 184 and 200 362 expired on March 28 2006 In the U S the patents covering the foundational PCR process expired on March 29 2005 This product is sold under licensing arrangements with the USB Corporation The purchase price of this product includes limited nontransferable rights under U S Patent Application Serial Number 11 171 008 owned by the USB Corporation to use only this amount of the product to practice the claims in said patent solely for activities of end users within the fields of life science research and forensic analysis of genetic material relating to or obtained as the result of criminal investigations or disaster sites conducted either by or for a governmental entity or for use
37. emElite ID 10X Primer Pair Mix StemElite ID 5X Enzyme Mix and Water Amplification Grade PCR is performed and ILS is added to the amplified product The alleles are resolved using capillary electrophoresis CE and CE data are analyzed using genotyping software and the parameters given on the Promega web site at www promega com stemeliteid This site provides instructions and applications to set the report parameters in the GeneMapper or GeneMapper ID software to make genotyping easier and more accurate This manual contains separate protocols for amplification of STR loci and detection of amplified products These protocols were tested at Promega For more information about required instrumentation and software see Section 3 DNA Sample Preparation Section 6 lt t Amplification Setup Section 7 A lt t Thermal Cycling Section 7 B GeneAmp PCR System 9700 GeneAmp PCR System 9600 Instrument Setup and Sample Preparation Applied Biosystems ABI PRISM 3100 or ABI PRISM 310 3130 or 3130x Genetic 3100 Avant Genetic Genetic Analyzer Analyzer Analyzer with Data Section 9 Section 8 Collection Software Version 2 0 Section 8 Data Analysis Section 10 GeneMapper ID Software GeneMapper Software Version 3 2 Version 4 0 Figure 2 An overview of the StemElite ID System protocol Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608
38. esis Instrument Preparation 12 9 Detection of Amplified Fragments Using the ABI PRISM 310 ez url n 14 A Capillary Electrophoresis Sample Preparation atro 14 B Capillary Electrophoresis Instrument Preparation 15 TU Datis Snaby SiS odio tmi ELE DEDE 17 As Downloading Panel and BI FeS otisses 17 B Importing Panel and Bin Files for GeneMapper ID and Gene Mapper SOLCHE cicius edited itd ups cc dcbet esie durus 17 C Creating a Method with GeneMapper ID and Gepe Mapper SOLD HEB edes itemtetlentntinedu uueitsa piene ritu ee 18 D Creating a nize andat essa teh irai re ne mtr eter mttaer 21 Fs MO D i pp 22 I CB qn i o etree Tene UR a bt nr eer etree tnt errr ert 2 COM S LM re ee rece tr Te 29 eB civi eo ee eer er 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 Printed in USA Part TM307 10 08 Page 1 Promega TE TS NOO NO aera D 27 A Afpliticatiorr nd Fragment ECCI ON us tusedsssensececsonnenuncatvcentieasstvtyseaveuniunn 27 B GeneMapper and GeneMapper ID Analysis Software 30 12 A DDOBO D ond ERR MEUSE N 33 A Additional STR Locus nfofrnallOD eoe rnt rrt ettet 33 P The Taber mal Line Sta an OU scaena itae rhe tir pri bob tbid 34 C Preparing the StemElite ID Syste
39. etic Analyzers These protocols were tested at Promega Corporation Amplification and detection instrumentation may vary You may need to optimize protocols including cycle number and injection time for each laboratory instrument Protocols for operation of the fluorescence detection instruments should be obtained from the instrument manufacturer Matrix standards are required for initial setup of the color separation matrix Section 4 The matrix standards are sold separately and are available for the ABI PRISM 310 Genetic Analyzer PowerPlex Matrix Standards 310 Cat DG4640 and the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xI Genetic Analyzers PowerPlex Matrix Standards 3100 3130 Cat DG4650 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 TM307 10 08 Page 5 4 Matrix Standardization or Spectral Calibration Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 310 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130x Genetic Analyzers A matrix must be generated for each individual instrument Very high peak heights may not be perfectly separated spectrally and an allele peak in one color channel can bleed into another color channel A poor or incorrect matrix will allow this a
40. f these settings have been optimized and are different from the recommended settings in the user bulletin Analysis Meth od Ed itor HID SJ i Peak Detector Peak Quality Quality Flags Bin Set StemElite ID Binz 1 0 Ww Use marker zpecific stutter ratio if available Marker Repeat Type Cutoff Value MinusA Ratio Minus Distance Minuz Stutter Ratio Minus Stutter Distance Plus Stutter Ratio Plus Stutter Distance Amelogenin Cutott Range Filter Factory Defaults Cancel 7816TA Figure 3 GeneMapper ID Analysis Method 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 TM307 10 08 Page 19 Analysis Method Editor Microsatellite General Allele Peak Detector Peak Quality Quality Flags Bin Set StemElite ID Bins 1 0 Marker Repeat Type Use marker specific stutter ratio if available Values for dinucleotide repeats are calculated automatically Mono i Tetra Cut off value PlusA ratio PlusA distance Stutter ratio Stutter distance Range Filter Factory Defaults 7817TA Figure 4 GeneMapper Analysis Method Allele tab 10 C Creating a Method with GeneMapper ID and GeneMapper Software continued 10 Select the Peak Detector tab We recommend the settings shown in Figure 5 Notes 1 Select full range or p
41. han the repeat length complicates interpretation and assignment of alleles There appears to be a correlation between a high degree of polymorphism a tendency for microvariants and increased mutation rate 14 15 Thus D21911 displays numerous relatively common microvariants CE related artifacts spikes are occasionally seen in one or all of the color channels 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 Reinject the samples to confirm Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM307 Printed in USA Page 26 10 08 11 Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail techserv promega com 11 A Amplification and Fragment Detection Symptoms Causes and Comments Faint or absent allele peaks Impure template DNA Depending on the DNA extraction procedure used and the sample source inhibitors may be present in the DNA sample Insufficient template Use the recommended amount of template DNA Incorrect amplification program Confirm the amplification program High salt concentration or altered pH If the DNA templat
42. heating block water bath or thermal cycler e 310 capillaries 47cm x 50pm e performance optimized polymer 4 POP 4 e glass syringe 1ml e 10X Genetic Analyzer buffer with EDTA e sample tubes and septa aerosol resistant pipette tips Hi Di formamide Applied Biosystems Cat 4311320 e PowerPlex Matrix Standards 310 Cat DG4640 e crushed ice or ice water bath The quality of the formamide is critical Use Hi Di formamide with a conductivity less than 100pS cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 10005 cm may contain ions that compete with DNA during injection This results in lower peak heights in the resulting data and reduced sensitivity A longer injection time may not increase the signal Caution Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 9 A Capillary Electrophoresis Sample Preparation 1 Prepare a loading cocktail by combining the Internal Lane Standard 600 ILS 600 and Hi Di formamide as follows 1 0p1 ILS 600 x injections 24 0pl Hi Di formamide x st injections Note The volume of internal lane standard used in the loading cocktail can be increased or
43. ilable at www promega com stemeliteid 10 C Creating a Method with GeneMapper ID and GeneMapper Software Select Tools then GeneMapper Manager Select the Analysis Methods tab Select New and a new analysis method dialog box will open Select HID if available or Microsatellite Select OK Enter a descriptive name for the analysis method such as StemElite ID Select the Allele tab QU OQ M EOS qr spa Select the bin set corresponding to the StemElite ID System StemElite ID Bins 1 0 Note If using GeneMapper analysis software instead of GeneMapper ID software use the custom bin generation tool in Section 10 B Step 9 above to generate a custom bin file 8 Ensure that the Use marker specific stutter ratio if available box is checked Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM307 Printed in USA Page 18 10 08 9 For proper filtering of peaks when using the StemElite ID System enter the values shown in Figure 3 when using GeneMapper ID and HID analysis methods or Figure 4 when using GeneMapper and Microsatellite MS analysis methods For an explanation of the proper usage and effect of these settings refer to the Applied Biosystems user bulletin titled Installation Procedures and New Features for GeneMapper ID Software 3 2 Note Some o
44. ination with another template DNA or of the color or all of the color channels channel of previously amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not completely denatured Heat denature the samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to loading the capillary Artifacts of STR amplification PCR amplification of STR systems sometimes generates artifacts that appear as peaks that are one repeat unit smaller than the allele and have low peak heights These stutter peaks can be high if the samples are overloaded 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 Reinject the samples to confirm CE related artifacts contaminants Contaminants in the water used with the instrument or to dilute the 10X Genetic Analyzer buffer may generate peaks in the blue and green dye colors Use autoclaved water change vials and wash buffer reservoir High background caused by excessive amount of DNA Use less template DNA or reduce the number of cycles in the amplification program by 2 4 cycles 10 20 or 10 18 cycling Heights of pull up or bleedthrough peaks are too high or a poor or incorrect matrix has been applied t
45. ls to prepare and use this internal lane standard are provided in Sections 8 and 9 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM307 Page 34 Printed in USA 10 08 120 140 160 180 225 250 275 325 350 375 425 450 475 Figure 9 An electropherogram showing the fragments of the Internal Lane Standard 600 12 C Preparing the StemElite ID System Master Mix A worksheet to calculate the required amount of each PCR master mix component is provided in Table 5 Multiply the volume pl per reaction by the total number of reactions to obtain the final master mix volume pl Table 5 Master Mix for the StemElite ID System PCR Master Volume Per Numberof _ Final Volume Mix Component Reaction Reactions ul StemElite D 5X Enzyme Mix 5 0pl x StemElite ID 10X Primer Pair Mix 2 51 x Water Amplification Grade pl x Per tube x template DNA volume 1 4ng up to17 5pl x total reaction volume 25ul x The master mix volume and template DNA volume should total 25pl Consider the volume of template DNA and add Water Amplification Grade to the master mix to bring the final volume of the final reaction to 25p1 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 i
46. m Master MiX ivssisisisspccasssisisinistinssnssiasn 35 D Composition of Buffers and Solutions s rtr dicet 36 ME ri TT RE TR T CNN M T 36 imide cH sce DEDQUPIS pocos identitate svn AE UR NM Bux 38 1 Description Cell line misidentification and cross contamination have become important concerns for researchers For example stem cell lines can become contaminated by the mouse cell line making up the feeder cell layers on which stem cells are propagated It has been shown that laboratories have invested substantial time and effort researching cell lines that were revealed to be misidentified 1 The situation has prompted the National Institutes of Health to issue a notice to researchers strongly recommending authentication procedures when using cultured cells 2 Genetic profiling can be used as a tool for stem cell line quality assurance and human cell lines can be authenticated using short tandem repeat STR loci 3 5 STR loci consist of short repetitive sequence elements 3 7 base pairs in length These repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers which can be detected using the polymerase chain reaction 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 Early efforts toward genetic characterizatio
47. me 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 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 TM307 Printed in USA Page 12 10 08 O Select Regular in the Type drop down list and select the run module you created in the previous step in the Run Module drop down list Lastly select F in the Dye Set drop down list Select OK 3 In the Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select GeneMapper Generic in the Application drop down list and select the appropriate plate type 96 well Add entries in the owner and operator windows and select OK Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions 4 In the GeneMapper plate record enter sample names in the appropriate cells Scroll to the right In the Results group 1 column select the desired results group In the Instrument Protocol 1 column select the protocol you created in Step 2 Be sure this information is present for each row that contains a sample name Select OK Note To create a new results group select New in the drop down menu in the results group column Select the General tab and enter a name Select the An
48. n USA Part TM307 10 08 Page 35 12 D Composition of Buffers and Solutions TE buffer 10mM Tris HCl 0 1mM EDTA pH 8 0 221g Tris base 0 037g EDTA Na EDTA 2H 0 Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCI Bring the final volume to 1 liter with deionized water 12 E References 1 Chatterjee R 2007 Cell biology Cases of mistaken identity Science 315 928 31 2 Ruiz Bravo N and Gottesman M 2007 Notice regarding authentication of cultured cell lines This can be viewed online at http grants nih gov grants guide notice files NOT OD 08 017 html 3 Yoshino K et al 2006 Essential role for gene profiling analysis in the authentication of human cell lines Human Cell 19 43 8 4 Szibor R et al 2003 Cell line DNA typing in forensic genetics the necessity of reliable standards Forensic Sci Int 138 37 43 5 Dirks W G et al 2005 Short tandem repeat DNA typing provides an international reference standard for authentication of human cell lines ALTEX 22 103 9 6 Masters J R et al 2001 Short tandem repeat profiling provides an international reference standard for human cell lines Proc Natl Acad Sci USA 98 8012 7 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 TM307 Printed in USA Page 36 10 08 7 2001
49. n of human cell lines used the PowerPlex 1 2 System Substantial databases derived using the eight STR loci and Amelogenin locus of the PowerPlex 1 2 System currently exist 6 7 To support those efforts we included the PowerPlex 1 2 System loci in the new StemElite ID System 9 plus additional loci The D21511 locus was included for additional discrimination power To provide a powerful and complete system for cell line identification we developed the StemElite ID System which contains all reagents required for successful identification and authentication of human stem cell lines and detection of cell line contaminants in a research laboratory Additionally we have incorporated a sensitive marker that specifically detects the presence of DNA from the mouse Mus musculus This locus has been developed to co amplify with the existing human loci in the primer set provided with this 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 TM307 Printed in USA Page 2 10 08 system The StemElite ID System offers the convenience of room temperature reaction assembly by including a hot start Tag DNA polymerase system which is included in a 5X enzyme mix This allows a simple one step addition of Taq DNA polymerase dNTPs MgCl and reaction buffer necessary for DNA amplification The StemElite TD System allows co
50. ne the results for the negative control The negative control should be devoid of amplification products 2 Examine the results for the 9947A positive control DNA Compare the control DNA allelic repeat sizes with the locus specific allelic ladder The expected 9947A DNA allele designations for each locus are listed in Table 2 Table 2 Expected Allele Designations for the 9947A DNA STR Locus Alleles D21S11 30 30 TH01 o dS TPOX 8 8 vWA 1718 Amelogenin X X CSF1PO 10 2 D165539 ILS D75820 10 11 D139317 11 11 D5S818 mN 10 H Results Representative results of the StemElite ID System are shown in Figure 6 The StemElite ID Allelic Ladder is shown in Figure 7 Locus to locus peak height imbalance will likely occur with cell line DNA Normal genomic DNA has equal copies of the loci and amplification will result in relatively even locus to locus balance Cell line DNA can have mutations that affect the locus to locus allele peak height balance The STR genotype of a cell line can evolve over multiple passages Users can genotype cell line DNA regularly with the StemElite ID System to monitor any change in the STR genotype Additionally cell lines have occasionally been observed to have tri allelic patterns at a locus 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 TM307 10 08 Page 23
51. nimize these 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 Printed in USA Part TM307 10 08 Page 25 10 H Results continued We have carefully selected STR loci and primers to avoid or minimize artifacts including those associated with Taq DNA polymerase such as repeat slippage and terminal nucleotide addition Repeat slippage 9 10 sometimes called n 4 peaks stutter or shadow bands is due to the loss of a repeat unit during DNA amplification somatic variation within the DNA sample material or both The amount of this artifact observed depends primarily on the locus and the DNA sequence being amplified Terminal nucleotide addition 11 12 occurs when Taq DNA polymerase adds a nucleotide generally adenine to the 3 ends of amplified DNA fragments in a template independent manner The efficiency 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 30 minutes 13 to the amplification protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used The presence of microvariant alleles alleles differing from one another by lengths other t
52. o the samples e Generate a new matrix or spectral calibration e Sensitivities of instruments may vary Optimize the injection conditions See Section 8 B or 9 B Long term storage of amplified sample in formamide can result in degradation Repeat preparation of samples using fresh formamide The CE polymer was beyond its expiration date or polymer was stored at room temperature for more than one week Maintain instrumentation on a daily or weekly basis as recommended by the manufacturer 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 TM307 Printed in USA Page 28 10 08 Symptoms Causes and Comments Precipitate observed in A precipitate may form as a result of thermodenaturation samples after amplification of the protein associated with hot start This precipitate does not affect downstream amplification or capillary performance Allelic ladder not running Be sure the allelic ladder and samples are from the same the same as the sample instrument run Poor injection of allelic ladder Include more than one ladder per instrument run Peak height imbalance Many cell lines will exhibit peak height imbalance after many passages This may be normal for some cell lines Excessive amount of DNA Amplification of too much template can result in an imbalance with yields with smaller loci showing more product than
53. old for 30 seconds ramp 50 seconds to 70 C hold for 45 seconds for 10 cycles then 90 C for 30 seconds then ramp 60 seconds to 60 C hold for 30 seconds ramp 100 to 94 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 60 C for 30 seconds ramp 23 to 70 C for 45 seconds for 22 cycles then ramp 50 seconds to 70 C hold for 45 seconds 60 C for 30 minutes for 22 cycles then 4 C soak 60 C for 30 minutes 4 C soak 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 TM307 Printed in USA Page 10 10 08 8 Detection of Amplified Fragments Using the Applied Biosystems 3130 or 3130x Genetic Analyzer and ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Versions 2 0 and 3 0 Materials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice or ice water bath aerosol resistant pipette tips e 2100 or 3130 capillary array 36cm e performance optimized polymer 4 POP 4 for the 3100 or 3130 e 10X Genetic Analyzer buffer with EDTA e MicroAmp optical 96 well plate and septa Hi Di formamide Applied Biosystems Cat 4311320 e DowerPlex Matrix Standards 3100 3130 Cat DG4650 The quality of the form
54. ould not be sheared and should be free of contaminating protein and salts Poor quality DNA may result in increased background or amplification failure Too much or too little DNA in the reaction can cause amplification failure which can be manifested in several ways complete lack of amplification of all loci or dropout of all or subsets of the alleles Commercially available DNA purification systems such as the MagneSil Genomic Fixed Tissue DNA Purification System Cat MD1490 Wizard SV Genomic DNA Purification System Cat A2360 Wizard Genomic DNA Purification System Cat A1620 and Maxwell 16 Cell LEV DNA Purification Kit Cat AS1140 produce DNA of sufficient quality for the StemElite ID System These systems yield clean DNA for STR analysis easily and efficiently The MagneSil Resin eliminates PCR inhibitors and contaminants frequently encountered in DNA samples DNA should be stored in a low EDTA TE buffer pH 8 0 We recommend TE buffer 10mM Tris HCl 0 1mM EDTA pH 8 0 High salt concentration or altered pH can affect amplification If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the DNA volume should not exceed 20 of the total reaction volume Carryover of K Na Mg or EDTA can negatively affect PCR 6 B DNA Quantitation We recommend quantitating the DNA prior to use in the StemElite ID System as either too much or too little DNA can cause rea
55. ry small amounts of nontemplate human DNA Extreme care should be taken to avoid cross contamination when preparing sample DNA handling primer pairs assembling amplification reactions and analyzing amplification products Reagents and materials used pre amplification StemElite ID 5X Enzyme Mix StemElite ID 10X Primer Pair Mix Amplification Grade Water and 9947A DNA are provided in a separate box and should be stored separately from those used post amplification StemElite ID Allelic Ladder and Internal Lane Standard 600 Always include a negative control reaction i e no template to detect reagent contamination We highly recommend the use of gloves and aerosol resistant pipette tips e g ART tips 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 TM307 Printed in USA Page 6 10 08 O Some of the 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 6 DNA Sample Preparation 6 4 DNA Purification DNA concentration purity and integrity are important considerations to ensure success with the StemElite ID System DNA sh
56. s well The PowerPlex Matrix Standards 310 Cat DG4640 is required for matrix standardization for the ABI PRISM 310 Genetic Analyzer For best results the PowerPlex Matrix Standards 3100 3130 Cat DG4650 should not be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 is required for spectral calibration on the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 and 3130xI Genetic Analyzers The PowerPlex Matrix Standards 310 Cat DG4640 cannot be used to generate a matrix on these instruments For protocols and additional information on matrix standardization see the PowerPlex Matrix Standards 310 Technical Bulletin TBD021 which is supplied with Cat DG4640 For protocols and additional information about spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 which is supplied with Cat DG4650 These manuals are available upon request from Promega or online at www promega com tbs 5 Precautions The quality of the purified DNA sample as well as small changes in buffers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can affect PCR success We suggest strict adherence to recommended procedures for amplification as well as electrophoresis and fluorescence detection PCR based STR analysis is subject to contamination by ve
57. sic laboratories HID analysis settings analyze data using panel and bin files which supply information regarding the alleles expected within a sample set An allelic ladder is used by the HID analysis algorithms to calculate offsets or variations in the migration of alleles on a particular instrument allowing the software to correct for subtle differences in sample migration caused by temperature voltage polymer and other factors Other versions of GeneMapper analysis software do not contain these options D Other versions of GeneMapper analysis software can be used to analyze StemElite D System data However laboratories using other versions must verify that all allelic ladders and positive controls are called correctly Panel and bin files must be created specifically for each CE instrument and are not interchangeable The GeneMapper software does not calculate offsets and subsequently the panel and bin files need to be customized to reflect the actual migration of fragments on a particular instrument A custom bin generation tool is available from Promega specifically to generate panel and bin files customized to reflect the migration of fragments on a particular instrument for the StemElite ID System This tool requires data from at least three analyzed runs of the StemElite ID Allelic Ladder which can be exported as a table as described on the Promega web site These instructions and the custom bin generation tool are ava
58. sis Instrument Preparation Refer to the instrument user s manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer Follow the manufacturer s recommendations for polymer storage and shelf life Polymer stored at room temperature for more than 1 week can result in broad or split peaks or extra peaks visible in one or all of the color channels Maintain the instrumentation on a daily or weekly basis as recommended by the manufacturer for optimal results and fewer instrument related artifacts Contaminants in the water used with the instrument or to dilute the 10X Genetic Analyzer buffer may generate peaks in the blue and green dye colors Use autoclaved water Analyze the 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 3130xI Genetic Analyzer with the following exceptions 1 In the Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36 POP4 in the Template drop down list Confirm that the injection time is 5 seconds and the injection voltage is 3kV Lengthen the run time to 2 000 seconds Give a descriptive name to your run module and select OK Note Sensitivities of instruments may vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection ti
59. the larger loci Use less template or reduce the number of cycles in the amplification program by 2 4 cycles Degraded DNA sample DNA template is degraded and the larger loci show diminished yield Repurify the template DNA Miscellaneous balance problems Thaw the StemElite ID 10X Primer Pair Mix completely and vortex for 15 seconds before using Do not centrifuge the 10X Primer Pair Mix after mixing Calibrate thermal cyclers and pipettes routinely Impure template DNA Inhibitors can lead to allele dropout or imbalance Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM307 10 08 Page 29 11 B GeneMapper and GeneMapper ID Analysis Software Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper ID software the analysis parameters and size standard must both have Basic or Advanced as the analysis type If they are different an error is obtained An insufficient number of ILS 600 fragments was defined Be sure to define at least one ILS 600 fragment smaller than the smallest sample peak and at least one ILS 600 fragment larger than the largest sample peak The internal lane standard was not properly identified in the sample Manually redefine the sizes of the size standard fragments in the sample Off ladder alleles An allelic ladder from

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