Thermo Scientific Canine Genotypes Panel 2.1
Contents
1. 1 Control DNA e negative control H 0 The total volume of the PCR reaction mix is enough to account for possible volume losses due to reagent pipetting A single 1 5 mL microcentrifuge tube and the above formulation can be used for up to 70 samples 2 Close the microcentrifuge tube and vortex at full speed for 5 s Spin the tube briefly to remove any liquid remaining in the cap 3 Label PCR reaction vessels and transfer 18 pL of the PCR reaction mix into each vessel 4 Add 2 pL of sample DNA extract or positive control DNA 0 25 ng pL into each vessel Allocate at least one vessel for a negative control and instead of DNA add 2 pL of H O into that vessel 5 Close the reaction vessels vortex gently and spin briefly to remove possible liquid from the caps or sealers 6 Immediately place the reaction vessels into a thermal cycler Start the PCR program Table 4 Thermal cycling programs of the Canine Genotypes Panel 2 1 for different PCR instruments PCR instrument Cycling profile senate settings Piko Thermal Cycler 1 98 C for 3 min Default settings Arktik Thermal Cycler 2 30 cycles of Eppendorf Mastercycler gradient 98 C for 15 s Eppendorf 60 C for 75 s Mastercycler pro S 72 C for 30 s SensoQuest LabCycler 3 72 C for 5 min 1 98 C for 3 min Ramping speed 100 2 30 cycles of 98 C for 15 s 60 C for 75 s 72 C for 30 s 3 72 C for 5 min ABI GeneAmp PCR System 9700 96 Wwell ABI Veriti2. 4 387 2 367 2 367 4 385 5 385 8 366 3 366 6 385 4 385 8 FH2088 123 9 127 8 123 3 127 2 122 6 126 5 123 7 1241 127 6 127 9 123 2 123 4 1271 1274 122 5 122 7 126 4 126 6 VWEX 158 5 158 5 158 2 158 2 155 4 155 4 158 3 158 6 158 3 158 6 158 1 158 3 158 1 158 3 155 3 155 5 155 3 155 5 FH2010 233 6 233 6 233 2 233 2 231 8 231 8 233 4 233 8 233 4 233 8 233 1 233 4 233 1 233 4 231 7 231 9 231 7 231 9 PEZ16 300 3 304 4 299 5 303 6 298 7 302 6 299 8 300 7 303 8 304 9 299 5 299 6 303 4 303 8 298 6 298 8 302 4 302 7 FH3313 414 4 419 9 413 7 419 1 413 7 4191 413 4 415 3 418 9 420 8 4 413 6 413 8 418 9 419 2 413 5 413 9 418 9 419 3 Analysis and interpretation of the results 11 Representative results The reagents and protocols of the Canine Genotypes Panel 2 1 have been optimized to deliver similar peak sizes within and between loci when applying an appropriate amount of high quality genomic DNA PCR and electrophoresis conditions are acceptable when the fluorescent intensities of the Canine Genotypes Control DNA alleles fall between 1000 and 4000 Relative Fluorescence Units RFU Variation within this range is acceptable and can occur due to specific performance character istics of the applied PCR or electrophoresis instruments We recommend optimizing both the DNA template amount fo
3. 8 202 2 213 9 214 3 201 7 201 9 213 8 214 0 199 5 199 8 212 0 212 1 FH2017 263 5 267 5 263 1 267 1 2621 2661 263 3 263 8 267 2 267 8 263 10 263 2 267 0 267 2 262 0 262 2 266 0 266 2 FH2309 394 7 394 7 394 9 394 9 395 0 395 0 394 5 394 9 394 5 394 9 394 9 395 0 394 9 395 0 894 8 395 1 394 8 395 1 PEZ05 103 103 102 7 102 7 101 6 101 6 102 8 103 2 102 8 103 2 102 5 102 8 102 5 102 8 101 2 101 9 101 2 101 9 FH2001 129 4 146 8 128 9 146 6 128 0 144 2 129 2 129 6 146 5 147 128 8 129 1 146 5 146 8 127 9 128 1 144 1 144 2 FH2328 171 1 206 2 170 6 205 7 167 9 203 7 170 8 171 4 205 8 206 6 170 5 170 7 205 6 205 8 167 8 168 0 203 6 203 8 FH2004 233 6 241 8 233 3 241 4 231 8 240 0 233 4 233 8 241 5 242 233 1 233 4 241 3 241 6 231 7 231 9 240 0 240 1 FH2361 345 5 347 4 344 9 346 9 3431 3451 345 2 345 7 347 2 347 6 344 7 3451 346 7 347 1 343 1 343 1 345 0 345 2 PEZ 21 89 1 971 89 2 97 0 87 8 95 6 88 9 89 3 96 9 97 3 89 0 89 3 96 8 97 1 87 4 88 3 95 2 96 0 FH2054 150 2 170 9 149 9 170 4 147 2 1677 150 150 4 170 7 171 1 149 8150 170 2 170 6 1471 1473 167 6 167 8 198 9 198 9 198 5 198 5 FH3377 196 2 196 2 198 6 199 2 198 6 199 2 198 4 198 6 198 4 198 6 196 1 196 4 196 2 196 4 FH2107 367 9 386 3 367 3 385 7 366 4 385 6 367 1 368 7 385
4. a background of human DNA Stutter peaks and allele calling Microsatellite amplification can result in one or more stutter peak arguably due to a phenomenon known as slipped strand mispairing Goldstein and Schl6tterer 1999 The stutter peaks typically lack one repeat unit relative to the true allele Hence for tetranucleotide repeat motifs they are typically 4 bp shorter than the true alleles A total of 16 markers of the Canine Genotypes Panel 2 1 are tetra nucleotide microsatellite loci Table 1 The PCR amplification of tetranucleotide short tandem repeat STR loci typically produces a minor product band 4 bp shorter than the corresponding main allele band PCR amplification results from tetranucleotide repeat loci are easier to interpret because only a single stutter band is typically observed in a position four bases shorter than allele band When interpreting the results it is noteworthy that within one locus the longer alleles may display smaller amplification yields peak sizes than the shorter alleles Moreover within some loci the longer alleles may display more significant stuttering than the shorter alleles A separate developmental validation study that assessed stutter percentages of Canine Genotypes Panel 2 1 kit has been conducted and published by M Dayton et al 2009 Plus A peaks Due to the proofreading activity 3 to 5 exonuclease activity of the Phusion Hot Start DNA Polymerase the Canine Genotyp
5. following into a 1 5 mL microcentrifuge tube e Number of samples x 11 pL of deionized formamide e Number of samples x 0 3 pL of GeneScan 500 LIZ Size Standard An excess volume to compensate for volume losses due to reagent pipetting is already included 2 Close the microcentrifuge tube and vortex it at full speed for 5 s Spin the tube briefly to collect liquid from the tube walls 3 Transfer 10 pL of the mix into each well of a 96 well plate compatible with the instrument 4 Add 1 5 pL of PCR product or PCR product diluted into H O see Electrophoresis into each well Mix the solutions by pipetting Seal the plate 5 Heat the plate at 95 C for 3 min to denature the samples and immediately chill the plate on ice crushed ice or ice water bath for at least 3 min A Place the plate in an auto sampler tray and close the instrument doors 7 Select the GeneScan 36_Pop4 module Use the following values for injection in combination with 36 cm capillaries e Inj Secs 10 0 e Inj kV 3 0 e Run kV 15 0 e Run C 60 e Run Time 1200 s 8 Begin electrophoresis according to the ABI PRISM User Guide instructions Electrophoresis Using ABI PRISM 3130 Genetic Analyzer or ABI PRISM 3130x Genetic Analyzer 1 Prepare a reaction mix for electrophoresis by combining the following into a 1 5 mL microcentrifuge tube e Number of samples x 11 pL of deionized formamide e Number of samples x 0 3 pL of GeneScan 500 LIZ S
6. trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Europe Customer Service cs molbio eu thermofisher com Technical Support ts molbio eu thermofisher com Tel 00800 222 00 888 Fax 00800 222 00 889 United States Customer Service cs molbio thermofisher com Technical Support ts molbio thermofisher com Tel 800 235 9880 Fax 800 292 6088 Canada Customer Service cs molbio thermofisher com Technical Support ts molbio thermofisher com Tel 800 340 9026 Fax 800 472 8322 Thermo SCIENTIFIC Part of Thermo Fisher Scientific
7. 3600 2400 1200 PEZS FH2001 FH2328 FH2004 FH2361 70 140 210 280 350 420 3600 150 190 194 eo 2400 368 97 1200 fe ever ee Greet se P PEZ21 FH2054 FH3377 FH2107 70 140 210 280 350 420 3600 2400 1200 FH2088 WFX FH2010 PEZ16 FH3313 B Canine Genotypes Panel 2 1 ge notyping profile of canine DNA extracted from buccal swab DNA was extracted using GeneJET Genomic DNA Purification Kit PEZ2 ZFX_Y PEZ_17 FH2017 70 140 210 280 350 420 2700 210 254 1800 107 123 202 900 161 15 391 395 FH2309 2700 1800 900 0 PEZS FH2001 FH2328 FH2004 FH2361 70 140 210 280 350 420 2700 1800 900 0 PEZI FH2054 FH3377 FH2107 70 140 210 280 350 420 2700 1800 4 132 389 900 128 q5g 70 H 292 377 230 300 0 FH2088 WFX FH2010 PEZI6 FH3313 C A mixed sample containing both canine and human DNA was genotyped using Canine Genotypes Panel 2 1 kit D A was extracted from a mixture of dog saliva and human blood using Thermo Scientific MagJET Genomic DNA Purification Kit 19 markers specific for dog 13 DNA were successfully amplified in
8. 96 Well Thermal Cycler Electrophoresis The Canine Genotypes Panel 2 1 has been optimized for electrophoresis using the ABI PRISM 3100 Genetic Analyzer ABI PRISM 3100 Avant Genetic Analyzer ABI PRISM 3130 Genetic Analyzer ABI PRISM 3130x Genetic Analyzer ABI PRISM 3500 Genetic Analyzer and ABI PRISM 3500x Genetic Analyzer all Applied Biosystems In addition to the instructions outlined below please refer to the instrument User Manual for electrophoresis details The Canine Genotypes Panel 2 1 is compatible with Filter Set GS requiring matrix files generated with the DS 33 Dye Primer Matrix Standard Set The matrix file values vary between instruments and electrophoresis conditions A matrix file must therefore be generated separately for each instrument The quantity of the microsatellite PCR products varies depending on the amount and quality of the DNA template used for the PCR reactions When you first start using the Canine Genotypes Panel 2 1 we strongly recommend preparing a dilution series of the PCR products and running electrophoresis in order to optimize the allele fluorescence intensities for the recommended range see Representative results For this experiment use undiluted PCR products and 1 5 1 10 1 20 and 1 40 PCR product dilutions in H O Electrophoresis Using ABI PRISM 3100 Avant Genetic Analyzer or ABI PRISM 3100 Genetic Analyzer 1 Prepare a reaction mix for electrophoresis by combining the
9. A TTCA 94 138 red vWF X 27 AGGAAT 151 187 red FH2010 24 ATGA 221 243 red PEZ16 27 GAAA 280 332 red FH3313 19 GAAA 340 446 red Dye colors are listed as they appear in electrophoresis with filter set G5 6FAM blue VIC green NED yellow PET red Size standard LIZ appears in orange color Kit components and storage conditions The Canine Genotypes Panel 2 1 kit contains all reagents necessary to co amplify 18 STR loci and one gender determination locus see Table 1 for locus descriptions Composition of the kit e Canine Genotypes Panel 2 1 Master Mix A PCR master mix in an optimized buffer containing MgCl deoxynucleoside triphosphates dATP dCTP dGTP and dTTP and Phusion Hot Start DNA Polymerase 0 05 U uL e Canine Genotypes Panel 2 1 Primer Mix An optimized PCR primer mix in a buffer including forward and reverse primers for the PEZ02 ZFX Y PEZ17 FH2017 FH2309 PEZOS FH2001 FH2328 FH2004 FH2361 PEZ21 FH2054 FH3377 FH2107 FH2088 vWEX FH2010 PEZ16 FH3313 loci One primer from each primer pair is end labeled with a fluorescent dye e Canine Genotypes Panel 2 1 Control DNA Canine Control DNA at 0 25 ng pL concentration is used for verification of PCR and electrophoresis conditions Genomic DNA is derived from ATCC MDCK 1 canine cell line All kit components should be stored at 20 C Repeated freezing and thawing of the components will affect the performance of the ki
10. Thermo Scientific Canine Genotypes Panel 2 1 F 864S 100 reactions an D o be a D w z D 5 o Product Description Parentage testing individual identification and investigation of forensic cases using short tandem repeat STR loci Short Tandem Repeat STR loci or microsatellites are a class of nuclear DNA markers consisting of tandemly repetitive sequence motifs of two to seven base pairs in length Alleles of STR loci vary by the number of times a given sequence motif is repeated STR alleles are detected using Polymerase Chain Reaction PCR and by separating the amplification products using electrophoresis Due to their high level of polymorphism and Mendelian inheritance microsatellites have become the markers of choice for parentage testing and individual identification The use of STRs for the characterization of dogs biological evidence became commonly used in forensic cases as well Canine Genotypes Panel 2 1 is an easy to use STR genotyping kit that has been validated for use in canine forensics see references The kit is compatible with genomic DNA extracted from various types of dog biological samples liquid or dried blood saliva stains buccal cells hair and blood saliva mixture samples Kit overview Thermo Scientific Canine Genotypes Panel 2 1 encompasses the following 19 loci PEZ02 ZFX Y PEZ17 FH2017 FH2309 PEZ05 FH2001 FH2328 FH2004 FH2361 PEZ21 FH2054 FH3377 FH2107 FH2088
11. ase providing the following features e Allele callings represent the true alleles of an individual instead of plus A peaks or split peaks typically encountered when using e g Taq DNA polymerase This is due to the proofreading activity of the Phusion Hot Start DNA Polymerase The results are not impaired by the tendency of non proofreading DNA polymerases to add an extra nucleotide most often adenine to the end of the amplification products e The high processivity of Phusion Hot Start DNA Polymerase allows for robust and high yield amplification of all target loci High processivity enables reliable amplification of even the longest fragments and avoids allele drop out occurrences during multiplex PCR which can present a problem with difficult templates and or low genomic DNA copy numbers when using a Tag DNA polymerase Table 1 Locus descriptions for the Canine Genotypes Panel 2 1 markers Locus Chromosome Repeat Motif Size range bp Dye color PEZ02 17 GGAA 104 145 blue ZFX Y X Y 159 164 blue PEZ17 4 GAAA 190 225 blue FH2017 15 AG GT AGAT GATA p 256 276 blue FH2309 1 GAAA 339 428 blue PEZ05 12 TTTA 92 117 green FH2001 23 GATA 118 160 green FH2328 33 GAAA 171 213 green FH2004 11 AAAG 232 326 green FH2361 29 GAAA 322 439 green PEZ21 2 AAAT 83 103 yellow FH2054 12 GATA 139 177 yellow FH3377 3 GAAAA 183 305 yellow FH2107 GAAA 291 426 yellow FH2088 15 TTT
12. e of the DS 33 Dye Primer Matrix Standard e POP 7 for ABI 3500 and POP 4 for others Performance Optimized Polymer Applied Biosystems e Deionized formamide e Genetic Analyzer vessels and septums Applied Biosystems e Additional electrophoresis consumables are required Please refer to the ABI PRISM User Guides for further details Samples and DNA extraction The Canine Genotypes Panel 2 1 has been optimized for use with dog blood hair cheek swab saliva stains and saliva blood mixed samples However use of high quality genomic DNA isolated from other tissue is possible The Canine Genotypes Panel 2 1 delivers optimal results when 1 2 ng of high quality genomic DNA is applied in PCR volume of 20 pL However the kit delivers acceptable results with genomic DNA amounts ranging from 0 25 ng to 10 ng Following these recommendation guidelines is important application of too little or too much template DNA can result in compromised amplification of some all microsatellites undesired overshoot of some all markers and or undesired occur rence of non specific amplification products DNA yield DNA purity and the amount of PCR inhibitors may vary due to different DNA extraction protocols When you first start using the Canine Geno types Panel 2 1 we strongly recommend preparing dilution series of the extracted DNA in order to optimize the amount of template DNA needed for PCR The Canine Genotypes Panel 2 1 delivers
13. e quite low compared to specific signals of amplified microsatellites attention should be paid when interpreting the results The nonspecific products might be produced following amplification in locus FH2361 328 and 407 bp FH3377 217 bp FH3313 409 bp FH2107 327 bp It is highly probable that these peaks are PCR artifacts generated during 19 plex PCR One could expect to see single peaks common across most genotypes which are always identical and do not cause data misinterpretation References 1 Dayton M et al 2009 Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Material Croatian Medical Journal vol 50 no 3 pp 268 285 2 Kanthaswamy S et al 2009 Canine Population Data Generated from a Multiplex STR Kit for Use in Forensic Casework Journal of Forensic Sciences vol 54 no 4 pp 829 840 3 Ogden R et al 2012 Genetic data from 15 STR loci for forensic individual identification and parentage analyses in UK domestic dogs Canis lupus familiaris Forensic Science International vol 6 no 2 pp e63 e65 Troubleshooting Problem Faint or no signals from the test and add sample DNA into PCR in DNA quantity of the test sample the quantity recommended in this is below the assay sensitivity Recommended Actions Measure the DNA concentration Instruction Manual sample for all loci but normal signals for all loci from the Cont
14. es Panel 2 1 results do not contain plus A peaks Therefore allele callings using the kit always represent the true alleles of an individual instead of the plus A peak typically interpreted when using e g a Taq DNA polymerase Species specificity The Canine Genotypes Panel 2 1 is specific to canine DNA and full 19 locus profiles were not observed while testing DNA samples from chicken mouse rat horse cow pig cat fish monkey and human Dayton et al 2009 Most peaks fall outside of the canine allele size ranges with a few peaks close to canine allele positions However none of the peaks exhibited in any of the non canine species appeared to exhibit the morphology of STR products all peaks above 500 RFU lack stutter peaks which is helpful for evaluation if non canine species amplifica tion has occurred in a DNA mixture Dayton et al 2009 The Canine Genotypes Panel 2 1 kit is also suitable for STR genotyping of wolf Canis lupus DNA The peaks obtained using wolf DNA are concordant with the allele spectrum of the domestic dog for each locus Dayton et al 2009 14 Note on possible nonspecific artifacts As determined by many independent amplifications of different dog DNA samples including mixed human dog DNA samples prepared using various DNA extraction methods including control DNA samples some constant nonspecific amplifications in particular loci were observed Although the signals from nonspecific products ar
15. high quality results when DNA purified with Thermo Scientific DNA purification kits is used see Table 3 Table 3 Thermo Scientific genomic DNA purification kits for different canine samples Sample type Recommended genomic DNA purification kit GeneJET Whole Blood Genomic DNA Purification Mini Kit Liquid blood EDTA citrate K0781 K0782 Dried blood spot MagJET Whole Blood Genomic DNA Kit K2741 K2742 MagJET Genomic DNA Kit Buccal K2721 K2722 Saliva stains GeneJET Genomic DNA Purification Kit Hair follicle K0721 KO722 MagJET Genomic DNA Kit Mixed samples of blood and saliva KO721 K2722 Note For more details about genomic DNA purification protocols please contact technical support Europe ts molbio eu thermofisher com or US ts molbio thermofisher com PCR The Canine Genotypes Panel 2 1 utilizes Phusion Hot Start DNA polymerase that is inactive at room temperature Nevertheless in order to maximize the specificity and uniformity of the amplification products and to minimize cross contaminating aerosols we strongly recommend that PCR reactions are always set up on ice 1 Prepare a reaction mix for PCR on ice by combining the following into a 1 5 mL microcentrifuge tube e Volume of Canine Genotypes Panel 2 1 Master Mix N x 10 pL e Volume of Canine Genotypes Panel 2 1 Primer Mix N x 10 pL N Number of samples Include the following controls e positive control Canine Genotypes Panel 2
16. ize Standard The formulas provide excess volume to collect liquid from the tube walls 2 Close the microcentrifuge tube and vortex it at full speed for 5 s Spin the tube briefly to remove possible liquid from the cap 3 Transfer 10 pL of the mix into each well of a 96 well plate compatible with the instrument 8 Add 1 pL of PCR product or PCR product diluted into H O see Electrophoresis into each well Mix the solutions by pipetting Seal the plate Heat the plate at 95 C for 3 min to denature the samples and immediately chill the plate on ice e g crushed ice or ice water bath for at least 3 min Place the plate in an auto sampler tray and close the instrument doors Select the Fragment Analysis 36_Pop4 module Use the following values for injection in combination with 36 cm capillaries e Inj Secs 12 e Inj kV 1 2 e Run kV 15 0 e Run C 60 e Run Time 1500 s Begin electrophoresis according to the ABI PRISM User Guide instructions 7 3 Electrophoresis Using ABI PRISM 3500 Genetic Analyzer or ABI PRISM 3500x Genetic Analyzer i Prepare a reaction mix for electrophoresis by combining the following into a 1 5 ml microcentrifuge tube e Number of samples x 11 pL of deionized formamide e Number of samples x 0 2 pL of GeneScan 500 LIZ Size Standard or GeneScan 600 LIZ Size Standard v2 0 The formulas provide excess volume to compensate for volume losses due to reagent pipetting Close the microcen
17. mized for PCR using the following thermal cyclers Eppendorf Mastercycler gradient Eppendorf ABI Veriti 96 Well Thermal Cycler Applied Biosystems ABI GeneAmp PCR System 9700 96 well Applied Biosystems Eppendorf Mastercycler pro S Eppendorf LabCycler SensoQuest Arktik Thermal Cycler and Piko Thermal Cycler Thermo Scientific Electrophoresis e Electrophoresis instrument The Canine Genotypes Panel 2 1 has been optimized for electrophoresis using the ABI PRISM 3100 Genetic Analyzer ABI PRISM 3100 Avant Genetic Analyzer ABI PRISM 3130 Genetic Analyzer ABI PRISM 3130xl Genetic Analyzer ABI 3500 Genetic Analyzer and ABI 3500x Genetic Analyzer all Applied Biosystems The use of Canine Genotypes Panel 2 1 in other genetic analyzers is likely to deliver similar results GeneScan 500 LIZ Size Standard Applied Biosystems The Canine Genotypes Panel 2 1 markers have been optimized for allele calling using the GeneScan 500 LIZ Size Standard e Note If you use the GeneScan 600 LIZ Size Standard v2 0 as an alternative please see Control DNA allelic size Table 5 for ABI 3500 Genetic Analyzer For other analyzers perform the appropriate optimization studies to support the use of this size standard with the Canine Genotypes Panel 2 1 kit e DS 33 Dye Primer Matrix Standard Set Applied Biosystems The end labeled primers of the Canine Genotypes Panel 2 1 kit are compatible with Filter Set G5 requiring the us
18. r PCR and the amount of PCR product dilutions used for electrophoresis so that the allele fluorescence intensities are between 1000 and 4000 RFU Peaks lower than 300 RFU and higher than 6000 RFU should be interpreted with caution Figures 1 A B and C show genotyping results with Canine Genotypes Panel 2 1 using 0 5 ng of Canine Control DNA DNA from buccal swab purified using GeneJET Genomic DNA Purification Kit and DNA from human blood dog saliva mixed sample purified using MagJET Genomic DNA Purification Kit respectively The PCR were carried out using Arktik Thermal Cycler and the amplification products were separated on an ABI PRISM 3130 xI Genetic Analyzer Figure 1 5700 3800 1900 PEZ2 ZFX_Y PEZ_17 FH2017 FH2309 80 160 240 320 400 5700 3800 1900 PEZ5 FH2001 FH2328 FH2004 FH2361 80 160 240 320 400 199 5700 3800 1900 PEZ21 FH2054 FH3377 FH2107 80 160 240 320 400 5700 3800 234 301 305 158 421 i800 124 128 45 FH2088 WRX FH2010 PEZ16 FH3313 A Canine Genotypes Panel 2 1 control DNA genotyping profile 12 70 140 210 280 350 420 3600 2400 1200 PEZ2 ZFX_Y PEZ_17 FH2017 FH2309
19. rol DNA DNA concentration of the test sample is too high or DNA purity and repeat the protocol Repeat DNA extraction procedure There has been a user error in Dilute the sample DNA into dH O e g 1 2 1 5 and 1 20 dilutions Faint or no signals from both the the PCR or electrophoresis Repeat the protocol Check if LIZ size standard is present test sample and the Control DNA for alld The cycling profile applied is not optimal for the Canine Genotypes Check the PCR program Overshoot for all or some loci and amplification products from the test sample but normal signals for all loci from the Control DNA f non specifi PEE ano SPEU The sample DNA quantity added into PCR is too high Measure the DNA concentration and add sample DNA into PCR in the quantity recommended in this Instruction Manual Alternatively repeat the protocol for a dilution series of the sample DNA into dH 0 e g 1 2 1 5 1 10 and 1 20 Control DNA dilutions Overshoot for all or some loci and There has been a user error in the a Repeat the protocol occurrence of non specific PCR or electrophoresis setup amplification products from both the test sample and the The cycling profile applied is not optimal for the Canine Genotypes Check the PCR program 15 Appendix I Avoiding carryover contamination Due to their high sensitivity PCR assays are susceptible to carryover contamination by previo
20. t and must be avoided The kit is stable for six months from the date of packaging when stored and handled properly The kit components and storage conditions are listed in Table 2 Table 2 Canine Genotypes Panel 2 1 kit components and storage conditions for F 864S sufficient for 100 reactions Kit Component Description Storage conditions Canine Genotypes Panel 2 1 toner Master Mix 1 tube blue cap 1 1 mL 20 C 20 C Store Canine Cones eMC 1 tube red cap 1 1 mL protected from light Primer Mix at all times Canine Genotypes Panel 2 1 non Control DNA 1 tube green cap 30 uL 20 C Repeated freezing and thawing of the components will affect the performance of the kit and must be avoided Important Note The primer mix should be protected from light all the times as fluorescent dyes in the primer mix are light sensitive Materials needed but not supplied Additional equipment and consumables required e DNA extraction can be performed using various methods The specific equipment and consumables recommended for DNA purification are listed in Samples and DNA extraction PCR e Water nuclease free e Disposable gloves e Microcentrifuge e Vortex e Pipettes e Aerosol resistant pipette tips e 1 5 ml microcentrifuge tubes e 0 2 ml PCR reaction vessels tubes and caps strips and strip caps or plates and plate sealers e Thermal Cycler The Canine Genotypes Panel 2 1 kit has been opti
21. transferable license under U S and foreign patents owned by BIO RAD Laboratories Inc to use is product No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of this product Use of this product is covered by US Patent No 6 127 155 The purchase of this product includes a limited non transferable immunity from suit under e foregoing patent claims for using only this amount of product for the purchaser s own internal research a ae o right under any other patent claim no right to perform any patented method and no right to perform commercial services of any kind including without imitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel is product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing icenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California thermoscientific com onebio 2014 Thermo Fisher Scientific Inc All rights reserved ABI PRISM Applied Biosystems GeneAmp GeneScan LIZ POP and Veriti are trademarks of Life Technologies and its subsidiaries Mastercycler is a trademark of Eppendorf AG and its subsidiaries Affibody is a trademark of Affibody Technology AB and its subsidiaries All other
22. trifuge tube and vortex it at full speed for 5 s Spin the tube briefly to collect liquid from the tube walls Transfer 10 pL of the mix into each well of a 96 well plate compatible with the instrument Add 1 pL of PCR product or PCR product diluted into H O see Electrophoresis into each well Mix the solutions by pipetting Seal the plate Heat the plate at 95 C for 3 min to denature the samples and immediately chill the plate on ice crushed ice or ice water bath for at least 3 min Place the plate in an auto sampler tray and close the instrument doors Select the Fragment Analysis 50_Pop7 module Use the following values for injection in combination with 50 cm capillaries e Inj Secs 8 Inj kV 1 5 e Run kV 19 5 e Run C 60 e Run Time 1330s Start electrophoresis according to the ABI PRISM User Guide instructions Table 5 Average allele sizes of all 19 loci for Canine control DNA using LIZ 500 size standard with ABI 3130x and ABI 3500 genetic analyzers and LIZ 600 size standard with ABI 3500 LIZ 500 LIZ 600 Allelei Allele2 Allele1 Allele2 Allele1 Allele2 PEZ02 130 9 130 9 130 5 130 5 429 5 429 5 130 7 131 2 130 7 131 2 130 4 130 7 130 4 130 7 129 4 129 6 129 4 129 6 ZEX Y 161 1 161 1 160 7 160 7 158 0 158 0 161 161 2 161 161 2 160 6 160 8 160 6 160 8 157 9 158 0 157 9 158 0 PEZ17 202 214 1 201 8 213 9 199 6 22 0 201
23. usly amplified PCR products A single molecule of amplified DNA may influence the results by contaminating the reaction mixture before PCR The following general guidelines should be followed in addition to other precautions mentioned in this Technical Manual in order to minimize the risk of carryover contamination D a 7 w w e Set up physically and strictly separate working places for 1 DNA extraction and sample preparation before PCR 2 setup of the PCR reactions and 3 preparing electrophoresis reagent mixes and performing electrophoresis Workflow in the laboratory should always be unidirectional from 1 to 3 and traffic from the electrophoresis working place to the other separated working places during the same day should be avoided e Use different laboratory equipment disposable gloves micropipettes pipette tip boxes laboratory coats etc in each working place e Change gloves frequently and always before leaving an area e Use aerosol resistant pipette tips e Use new and or sterilized glassware and plasticware Product use limitation is product has been developed and is sold exclusively for research purposes and in vitro use only This product has not been tested for use in agnostics or drug development nor is it suitable for administration to humans or animals License Information his product is sold under license from Affibody AB Sweden urchase price of this product includes a limited non
24. vWEX FH2010 PEZ16 FH3313 Table 1 These markers were developed and validated for use in forensics see references Validation study assessed both the robustness and reliability of the markers and the sensitivity and reproducibility of multiplex PCR assay The Canine Genotypes Panel 2 1 allows co amplification of the above markers in a single multiplex PCR reaction One primer from each primer pair is end labeled with a fluorescent Thermo dye Following PCR the fragments are separated and detected SCIENTIFIC in a single electrophoresis injection using an automated electrophoresis instrument such as ABI PRISM 3130 Genetic Analyzer ABI PRISM 3130x Genetic Analyzer ABI PRISM 3500 Genetic Analyzer or ABI PRISM 3500x Genetic Analyzer all Applied Biosystems The Canine Genotypes Panel 2 1 provides all of the reagents necessary for amplification of the 19 loci In addition the kit includes canine control DNA originating from an MDCK 1 cell line for verification of PCR and electrophoresis conditions The Canine Genotypes Panel 2 1 delivers optimal results when 1 2 ng of high quality genomic DNA is applied in PCR reaction volume of 20 pL The reagents and reaction protocols of the Canine Genotypes Panel 2 1 have been optimized to deliver similar amplification yields peak sizes for alleles within and between loci when an appropriate amount of high quality DNA is applied The kit employs Thermo Scientific Phusion Hot Start DNA Polymer
Download Pdf Manuals
Related Search