HID EVOlution - Thermo Fisher Scientific
Contents
1. qPCR STR Setup System Getting Started Guide except that you Import the converted 7500 results files as described below Import the converted 7500 results files D IMPORTANT The qPCR STR system sets up STR PCR reactions with one AmpF STR kit per run We recommend that you view your qPCR results in the SDS software then determine how you will batch samples for amplification with the appropriate AmpF STR kit 1 When you are prompted to import the 7500 results import either the converted human or male 7500 results files based on the AmpF STR kit that you selected for use with the samples 2 Review the information in the Sample Normalization Adjustment window a If necessary adjust the Req Amt ng to increase or decrease the target DNA input amount added during reaction setup b If you want to include samples for STR setup that are not automatically selected e If necessary adjust the Req Amt ng to increase or decrease the target DNA input amount added during reaction setup Select the Process check box Note For samples with undetermined quantification results in the imported 7500 results file s the system adds the maximum volume of DNA sample per reaction You cannot increase or decrease the volume of DNA sample added to the reaction by changing the value in the Req Amt ng column Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software 15 Quantifiler Du2. Section 2 Validation of Quantifiler Duo Kit on HID EVOlution qPCR STR Setup System Experiments and results Freedom EVOware Software v1 4 Figure 6 Quantification results obtained from the SRY target male in the qPCR reaction plates prepared using QuantiflerDuo plate script from DNA source plate Duo Male plate v1 4 Day 1 2 and 3 100 10 0 1 Log10 of Quantity ng pL 0 01 R u ciun O2xuoco u c u 0 iuoc u cu cf uocc DNA Concentration ng L uum ah ass sinh nm ai o o e Day D m Figure 7 Quantification results obtained from the RPPH1 target total human in the qPCR reaction plates prepared using QuantiflerDuo tubes script from DNA source tubes Duo Human tubes v1 4 Day 1 2 and 3 100 Lem E 10 o gt E 5 1 Qe o o ci 8 0 1 0 01 DNA Concentration ng L ARSE SW VY SR 558 928 33558 VVAR Ss Ss So Day T N ea Quantifiler Duo DNA Quantification Kit User Bulletin 23 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Experiments and results Freedom EVOware Software v1 4 Figure 8 Quantification results obtained from the SRY target male in the qPCR reaction plates prepared using QuantiflerDuo tubes script from DNA source tubes Duo Male tubes v1 4 Day 1 2 and 3 100 M a 10 2 w gt 1 o 0 1 0 01 DNA Concentration ng L 256 928 S258 979 ASST VSR Day N m
3. e QuantifilerDuo tubes page 10 page 9 The source plate Figure 2 on page 19 and the source tubes Figure 3 on page 19 were then run on each system on three consecutive days to create six qPCR reaction plates per system Figure 4 During each qPCR reaction setup run a fresh standard dilution series of the Quantifiler Duo DNA Standard was prepared on the qPCR STR system Figure 4 Precision and reproducibility studies qPCR reaction plate setup runs 20 Quantifiler9 Duo DNA Quantification Kit User Bulletin Studies Precision and reproducibility studies Contamination studies Normalization table verification studies Section 2 Validation of Quantifiler Duo Kit on HID EVOlution qPCR STR Setup System Experiments and results Freedom EVOware Software v1 4 On three consecutive days the two qPCR reaction plates generated from each qPCR setup run Figure 4 on page 20 were run on the 7500 Real Time PCR system six runs total per system Standard curve slope R2 and sample DNA concentration ranges were evaluated for the Ribonuclease P RNA Component H1 target RPPH1 total human and Sex determining Region Y target SRY male Cy values from the precision and reproducibility studies were evaluated Female samples with Cy values 40 for the Quantifiler Duo Male target and TE buffer blanks with Cy values 40 were manually set up for amplification using the MiniFiler PCR Amplification Kit or the Yfiler PCR Amplificatio
4. 1 1 AU0J pue sjd j25 M N uoneprjeA pue j0ddng y uonesynueng YNG ong g4JejJnueng Table 2 Reagent volumes for qPCR reaction setup with the Quantifiler Duo DNA Quantification Kit 25 uL reaction volume 2 Calculate the required volume of Quantifiler Duo kit reagents using Table 2 D IMPORTANT To obtain the required volume of primer mix you can place up to 3 tubes of primer mix in the reagent block for one run If you divide the minimum required volume of primer mix across 2 or3 tubes each tube must contain a multiple of 190 uL plus an additional 50 uL per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions Refer to About minimum required reagent volumes on page 25 of the HID EVOlution qPCR STR Setup System Getting Started Guide e Note The volumes in the table include the excess volume required per sample and per run necessary to compensate for evaporation and pipetting losses during the run Reagent Available Required Extracted Diluted Required Required Minimum required volume for volume in volume per DNA standard excess dead 80 extracted DNA samples and full tube of reaction samples samples reactions volumet 16 diluted standards reagent per run per run per run per tube Ax B C DI E A B c D E Quantifiler Duo Primer 1 4 mL 11 55 uL up to 80 16 3 50 uL 11948 uL in one tube Mix Quantifiler Duo PCR 5 mL 13 75 uL up to 80 16 3 50 uL 141211 uL in one tub
5. 90000000 00000000 Site number 2 o o o o o o o o o o o o o o o ooooooooo Oooooooooo 7 OOd Tm Site number 3 E 10 ERES oo TOOOOO Oooooooooo Oooooooooo 00000000 00000000 o o o o o o o o 00000000 00000000 1 S82 83 84 S5 Grid numbers 123 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 bg Note Locations for DNA samples in both a plate and tubes are shown but only one type of plasticware for extracted DNA can be placed on the workstation for a given run 1 2 200 pL disposable pipette tips DiTis 3 6 50 uL DiTis 7 Not used with the Quantifiler Duo kit Trough for T gEg buffer used with Quantifiler Human and Human Male DNA Quantification Kits only 8 Chilled qPCR Reagent Block 9 MicroAmp Optical 96 Well Reaction Plate qPCR reaction plate with 96 well metal plate adapter 10 MicroAmp Optical 96 Well Reaction Plate if extracted DNA samples are in a plate with 96 well metal plate adapter 11 Tube racks S1 through S5 for DNA sample tubes if extracted DNA samples are in tubes 10 Quantifiler9 Duo DNA Quantification Kit User Bulletin Section 1 Using the Quantifiler Duo DNA Quantification Kit with the HID EVOlution qPCR STR Setup System Run automated qPCR reaction setup Run automated qPCR reaction setup D IMPORTANT For all Quantifiler kits you can save time by creating a qPCR STR Sample file to
6. LICENSE Use of the Quantifiler Duo DNA Quantification Kit product is covered by US patent claims and patent claims outside the US The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product solely in forensic and paternity testing including reporting results of purchaser s activities for a fee or other commercial consideration and also for the purchaser s own internal research No right under any other patent claims such as apparatus or system claims for real time PCR is conveyed expressly by implication or by estoppel Further information on purchasing licenses may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Trademarks of Life Technologies Corporation and its affiliates include Applied Biosystems AB Design 9 AB Logo AmpF STR Cofiler FAM GeneAmp GeneMapper Identifiler MicroAmp MiniFiler NED PrepFiler Profiler Plus Quantifiler SEfiler Plus SGM Plus VICO and Yfiler Freedom EVO and Freedom EVOware are registered trademarks and HID EVOlution is a trademark of Tecan Group Ltd All other trademarks are the sole property of their respective owners 2010 Life Technologies Corporation All rights reserved 444527515 Part Number 4445275 Rev B 09 2010 Stock Number 112UB29 01 y applied Applied Biosystems Technic
7. Section 1 Using the Quantifiler Duo DNA Quantification Kit with the HID EVOlution qPCR STR Setup System Workflow for the Quantifiler9 Duo DNA Quantification Kit Workflow for the Quantifiler Duo DNA Quantification Kit To use the Quantifiler Duo kit on the qPCR STR system follow the instructions in the HID EVOlution qPCR STR Setup System Getting Started Guide PN 4426903 with the added and modified procedures listed in Table 1 and explained on pages 4 through 15 of this user bulletin M Note Figure 1 on page 5 illustrates the files imported and exported throughout the automated HID workflow Table 1 Workflow for using the Quantifiler Duo kit on the qPCR STR system To perform this workflow step Follow the procedures in One time tasks Perform one time tasks a Download and install Quantifiler Duo scripts and Converter software on page 4 b Create detectors for the 7500 Real Time PCR System on page 5 c If you have not already done so perform the other one time tasks described in the Getting Started Guide GSG Chapter 2 Pre Run Procedures qPCR reaction setup and qPCR 1 Perform routine maintenance and other pre run procedures GSG Chapter 2 Pre Run Procedures 2 Prepare qPCR reagents and labware a Determine required reagent volumes on page 6 b Place reagents in the qPCR reagent block on page 8 c Set up the samples reagents
8. TE Blank 1 nguL TE Blank TE Blank 60 ng uL 1 ngul 9 1 ngul B Cc D E F G H TE Blank 50 ng uL TE Blank 5 ng uL TE Blank 1 ng uL TE Blank 50 ng uL 1ng uL 0 1 ng uL 50 ng uL TE Blank 5 ng uL TE Blank TTE Blank TE Blank 10 ng uL TE Blank 0 1 ng uL TE Blank 1 t ng uL TE Blank 10 ng uL TE Blank 0 1 ng uL lo ngul Te Blank 2 ngu TE Blank 0 8 ng uL TE Blank TE Blank 5 nguL 0 5 ng uL 0 025 ngru TE Blank 0 5 ng uL TE Blank 5 ngul 6 8 ng ut 9 025 ng ut TE Blank TE Blank TE Blank 0 5 ng uL Figure 3 Sample tubes 1 5 mL set up with extracted DNA source tubes Female Male i 1 2 3 5 1 2 3 50 ng uL TE Blank 1 ng uL TEBlank 0 5ng uL 4 5 0 ng uL TE Blank 0 5 ng uL TE Blank 0 1 ng uL 6 TE Blank TE Blank 0 1 ng uL 7 10 ng uL TE Blank 0 5 ng uL TE Blank 0 025 ng uL 8 TE Blank TE Blank 0 025 ng uL 9 TE Blank 50 ng uL TE Blank 10 11 12 13 14 15 16 Quantifiler9 Duo DNA Quantification Kit User Bulletin 19 12 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Materials and methods qPCR reaction plate setup The following scripts worktable layouts and Quantifiler Duo reagent block configuration were used System Setup Script Worktable layout Reagent am EK configuration qPCR STR system qPCR e QuantifilerDuo plate See Table 4 on See Table 3 on
9. and plasticware on the worktable on page 10 3 Run automated qPCR reaction setup GSG Chapter 4 Run Automated qPCR Setup except that you use the scripts on page 11 4 Perform qPCR and review results a Convert and transfer the 7500 setup file on page 11 b Import the converted 7500 setup file and perform qPCR on page 12 c Analyze and review the qPCR results on page 13 d Export and convert the 7500 results files on page 14 STR PCR reaction setup 1 Prepare STR PCR reagents and plasticware GSG Chapter 6 Prepare STR PCR Reagents and Labware 2 Run automated STR PCR reaction setup GSG Chapter 7 Run Automated STR PCR Setup except that you use the instructions for importing qPCR results on page 15 t HID EVOlution gPCR STR Setup System Getting Started Guide PN 4426903 8 The converted 7500 setup file is imported as a SDS plate record Quantifiler9 Duo DNA Quantification Kit User Bulletin Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software One time tasks One time tasks Download and install Quantifiler Duo scripts and Converter software D IMPORTANT To use the Quantifiler Duo kit on the qPCR STR system you must download and install the e HID EVOlution Quantifiler Duo Converter Software v1 0 e HID EVOlution Quantifiler Duo Scripts About the The HID EVOlution Quantifiler Duo Converter Softwar
10. import sample information into the qPCR STR system software If you enter sample information manually for qPCR reaction setup the information is not retained and you will need to manually re enter the information for STR PCR reaction setup Refer to Chapter 2 of the HID EVOlution qPCR STR Setup System Getting Started Guide for more information on qPCR STR Sample files To run a script follow the instructions in Chapter 4 of the HID EVOlution qPCR STR Setup System Getting Started Guide using the appropriate Quantifiler Duo script from the following table Freedom EVOware software script selection for qPCR reaction setup 2 Maximum number If you arenan the ang the sample DRAIS Use the script of extracted DNA kit s in samples per run Quantifiler Duo Kit a 96 well plate QuantifilerDuo_plate esc 80 1 5 mL microcentrifuge QuantifilerDuo_tubes esc 80 tubes Perform qPCR amplification and review results Convert and transfer the 7500 setup file IMPORTANT After qPCR Reaction setup Quantifiler Duo kit only you must use the HID EVOlution Quantifiler Duo Converter Software to convert the 7500 setup file ReactionPlate1 txt or lt barcode gt txt generated during the qPCR reaction setup run This creates an importable plate record containing run parameters including detectors needed to run the Quantifiler Duo kit assay on the 7500 instrument 1 On the qPCR STR system navigate to
11. the C HIDEVOlution_qPCRSTRfiles folder then locate the file ReactionPlate1 txt or lt barcode gt txt 2 Optional Rename the file 3 Copy and transfer the ReactionPlatel txt or lt barcode gt txt file to a location where it can be accessed by the HID EVOlution Quantifiler Duo Converter Software Quantifiler9 Duo DNA Quantification Kit User Bulletin 11 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Perform qPCR amplification and review results 4 Convert the file a Start the Converter b Select Convert Quantifiler Duo 7500 Setup file then follow the prompts in the software N Note Refer to the HID EVOlution gPCR STR Setup System HID EVOlution Combination System Quantifiler amp Duo Converter Manual Tecan Document ID 396042 for details 5 Copy and transfer the converted 7500 setup file to a location where it can be accessed by the SDS software computer Import the converted 7500 setup file and perform qPCR 1 Start the 7500 Real Time PCR System and SDS Software a Start the computer b Power on the 7500 instrument c Start the SDS Software Note For more information on starting the 7500 Real Time PCR System and software refer to the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide 2 Load the qPCR reaction plate into the 7500 instrument refer to the Quantifiler Duo DNA Quan
12. v2 1 Contamination studies demonstrated clean liquid handling capabilities for the qPCR setup on EVOware Software v1 4 and v2 1 Normalization and STR setup studies confirm that the new Quantifiler Duo scripts and Quantifiler Duo converter provide accurate and reliable data handling for normalization and STR setup The stability study confirms that the Quantifiler Duo scripts prepare diluted standards that meet the performance requirements when stored according to the conditions recommended in the Quantifiler Duo DNA Quantification Kit User Guide Quantifiler9 Duo DNA Quantification Kit User Bulletin 33 For Research Forensic or Paternity Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES NOTICE TO PURCHASER LIMITED
13. 24 Quantifiler9 Duo DNA Quantification Kit User Bulletin Section 2 Validation of Quantifiler Duo Kit on HID EVOlution qPCR STR Setup System Experiments and results Freedom EVOware Software v1 4 Table 6 RPPH1 target human DNA standard deviations and concentrations Male Standard Deviations Sample Expected n Plate 1 Plate 2 Plate 3 n Tubes 1 Tubes 2 Tubes 3 ng pL for for Plate Tubes 0 025 4 0 009 0 022 0 010 4 0 028 0 009 0 005 0 1 4 0 009 0 024 0 021 2t N A N At N At 0 5 4 0 018 0 008 0 052 4 0 025 0 018 0 052 1 4 0 033 0 023 0 069 4 0 066 0 123 0 147 2 4 0 079 0 133 0 162 4 0 048 0 192 0 255 5 4 0 351 0 235 0 331 4 0 595 0 574 0 656 10 4 0 311 0 843 0 849 4 0 598 0 541 0 346 50 4 3 949 1 505 1 488 4 0 888 1 386 3 261 Female Concentration ng pL Sample Expected n for Plate 1 Plate 2 Plate 3 n for Tubes 1 Tubes 2 Tubes 3 ng uL Plate Tubes 0 5 2 0 35 0 32 0 31 0 32 0 45 0 46 2 0 46 0 42 0 46 0 46 0 60 0 55 1 2 0 76 0 67 0 64 0 63 0 92 0 85 2 0 99 0 86 0 98 0 89 1 08 1 12 2 2 1 53 1 54 1 19 1 46 1 85 1 87 2 1 82 1 82 1 63 1 84 2 05 2 04 5 2 4 12 4 50 3 44 3 86 4 88 4 94 2 4 37 4 55 4 66 4 38 5 43 5 45 10 2 8 98 9 92 7 05 7 70 9 63 2 9 48 10 59 1232 10 15 10 74 10 48 11 85 50 2 59 86 42 85 53 61 2 54 92 57 46 64 06 64 48 46 47 54 78 58 79 58 41 65 04 i Due to the num
14. 3 18 0 998 3 28 0998 3 42 0 995 2 3 19 0 998 3 22 0 996 3 24 0 997 3 37 0 994 3 3 24 0 998 3 39 0 992 3 29 0 998 3 39 0 997 Avg 3 15 0 998 3 26 0 995 3 27 0 998 3 39 0 995 Quantifiler Duo DNA Quantification Kit User Bulletin 21 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Experiments and results Freedom EVOware Software v1 4 DNA concentration The range of variation in the quantification results is e Comparable to the results seen during developmental validation reported in the Quantifiler Duo PCR Amplification User Guide at all tested concentrations of DNA among replicates and across three runs e Similar for source plate and source tubes DNA concentrations from the three runs for the source plate and tubes are summarized in Figures 5 through 8 on pages 22 and 24 The standard deviation values and concentration ranges are summarized in Table 6 on page 25 and Table 7 on page 26 Figure 5 Quantification results obtained from the RPPH1 target total human qPCR reaction plates prepared using QuantiflerDuo plate script from DNA source plate Duo Human plate v1 4 Day 1 2 and 3 100 Len T 10 o z w t ded i 1 tu o e ri 8 0 1 0 01 DNA Concentration ng L HGS SS gjg Tnne GARB SeR S 2 So Day 5t e 9 22 Quantifiler9 Duo DNA Quantification Kit User Bulletin
15. al Resources and Support biosystems 850 Lincoln Centre Drive Foster City CA 94404 USA For the latest technical resources and support information Phone 650 638 5800 Toll Free 800 345 5224 for all locations please refer to our Web site at P part of lik technologies www appliedbiosystems com www appliedbiosystems com support
16. applied biosystems USER BULLETIN SEPTEMBER 2010 HID EVOlution qPCR STR Setup System SUBJECT Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Purpose of this user bulletin The Quantifiler Duo DNA Quantification Kit Quantifiler Duo kit is now supported for automated qPCR reaction setup on the HID EVOlution qPCR STR Setup System qPCR STR system This user bulletin Provides Quantifiler Duo kit specific instructions to supplement the procedures in the HID EVOlution qPCR STR Setup System Getting Started Guide PN 4426903 e Describes the validation and verification studies of quantitative PCR qPCR setup on the qPCR STR system using the new Quantifiler Duo scripts and the Quantifiler Duo Converter Software v1 0 In this user bulletin This user bulletin covers Using the Quantifiler Duo DNA Quantification Kit with the HID EVOlution qPCR STR Setup Systemi raseri cases iem et eere ende 2 Validation of Quantifiler Duo Kit on HID EVOlution qPCR STR Setup DY SUSI P M 16 Prerequisites This user bulletin assumes that You have access to the following documents HID EVOlution qPCR STR Setup System Getting Started Guide PN 4426903 Quantifiler Duo DNA Quantification Kit User s Guide PN 4391294 Tecan HID EVOlution gPCR STR Setup System Application Manual Tecan Freedom EVO Op
17. ber of wells on the qPCR reaction plate only two samples were run Standard deviation not calculated Quantifiler9 Duo DNA Quantification Kit User Bulletin 25 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Experiments and results Freedom EVOware Software v1 4 Table 7 SRY target male DNA standard deviations Male Standard Deviations Sample Expected n Plate 1 Plate 2 Plate 3 n Tubes 1 Tubes 2 Tubes 3 ng pL for for Plate Tubes 0 025 4 0 007 0 019 0 023 4 0 011 0 011 0 013 0 1 4 0 010 0 012 0 022 21 N At N At N At 0 5 4 0 029 0 016 0 031 4 0 039 0 021 0 066 1 4 0 037 0 063 0 066 4 0 059 0 091 0 180 2 4 0 141 0 041 0 107 4 0 090 0 133 0 289 5 4 0 263 0 399 0 448 4 0 425 0 624 0 873 10 4 0 275 0 601 0 958 4 0 738 0 737 1 188 50 4 5 385 0 948 0 615 4 5 619 1 597 2 549 f Due to the number of wells on the qPCR reaction plate only two samples were run Standard deviation not calculated Contamination study Experiment Results 26 Cy values from the precision and reproducibility studies were evaluated The results of the contamination study are summarized in Table 8 Five TE buffer blanks exhibited CT values 40 and were manually set up for amplification using the MiniFiler PCR Amplification Kit None of these 5 TE buffer blanks exhibited STR profiles One female sample exhibited a Cy value of 37 97 for
18. conds If you are using a previously prepared DNA standard dilution series vortex and briefly centrifuge the DNA standard dilutions at low speed before use Place the labeled empty tubes or labeled pre prepared DNA standard dilution series in positions 1 through 8 as shown in Table 3 on page 9 D IMPORTANT Make sure to place each concentration in the correct location in the reagent block as shown in the orange table in Table 3 on page 9 Place the reagents in positions 9 through 15 as shown in Table 3 on page 9 Quantifiler9 Duo DNA Quantification Kit User Bulletin Section 1 Using the Quantifiler Duo DNA Quantification Kit with the HID EVOlution qPCR STR Setup System Prepare qPCR reagents and labware Table 3 Reagent block configuration for Quantifiler9 Duo kit Legend 1 8 1 5 mL tubes of pre prepared DNA standard dilution series arrange concentrations as shown in the corresponding orange table OR 1 5 mL empty tubes for DNA standard dilution series 9 11 Up to three 1 5 mL tube s of Quantifiler9 Duo Primer Mix Place the first tube in position 9 If you use more than one tube continue to position 10 then position 11 4 12 Quantifiler Duo PCR Reaction mix 13 Empty 5 mL VWR tube VWR part number 890005 596 for the master mix 14 If the run includes preparation of DNA standard dilution series Quantifiler Duo DNA Standard tube 15 If the run includes preparation of DNA standard dilution series Quan
19. d and Quantifiler Duo Dilution Buffer Required volumes for qPCR STR system prepared DNA standard dilution series Reagent Available volume in Minimum volumet full tube of reagent required on worktable Undiluted Quantifiler 120 uL 75 uL Duo DNA Standard Quantifiler Duo Dilution 1 8 mL 825 uL Buffer i Includes 50 uL dead volume per tube necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips meets the following three requirements The series was prepared in the last two weeks Usea pre prepared DNA standard dilution series if the pre prepared series The series was prepared according to the procedures specified in the Quantifiler Duo DNA Quantification Kit User s Manual or was prepared by the qPCR STR system Atleast 30 uL of each of the eight concentrations of diluted standard is available For reliable liquid detection it is recommended to use at least 50 uL of each of the eight concentrations of diluted standard 16 DNA standard dilution series samples Note Two replicates of the eight concentrations in the DNA standard dilution series are transferred to the qPCR reaction plate for a total of 30 uL includes the 4 uL necessary to prepare the two replicates of each concentration in the DNA standard dilution series plus the minimum required overfill volume Quantifiler9 Duo DNA Quantification Kit User Bulletin 8JeMjJOS
20. e Reaction Mix i A 50 uL dead volume per tube is necessary to ensure that the pipette tips remain submerged during aspiration so that liquid not air enters the tips 8 11 55 pL reactionx 80 16 3 reactions 50 uL tube 1193 45 uL in one tube If you divide the minimum volume of Primer Mix across multiple tubes each tube must contain a multiple of 190 uL plus an additional 50 uL per tube to ensure that the instrument detects adequate volume to prepare the selected number of reactions For example place at least 1000 uL in the first tube and 240 uL in the second tube 13 75 uL reactionx 80 16 3 reactions 50 pL tube 1411 25 pL in one tube asemge pue sjuebeaJ YJdb asedaly ujejss dmes Y1S YOdb uonnjoA3 CIH 24 YUM yy uonexunuenp YNA ong gJerynuenp eu fuis uon es Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Prepare qPCR reagents and labware Place reagents in the qPCR reagent block Use the following procedure to place the Quantifiler Duo kit reagents into the correct positions in the chilled qPCR reagent block 1 Thaw the reagents then prepare according to the Quantifile Duo DNA Quantification Kit User s Manual Thaw the primer mix completely then vortex 3 to 5 seconds and centrifuge briefly before opening the tube Swirl the PCR reaction mix gently before using Do not vortex it e Vortex the DNA standard 3 to 5 se
21. e ensures that the Quantifiler Duo kit Quantifiler Duo assay sample data is handled correctly by the SDS and qPCR STR Converter Software system software Use the Converter to Convert the 7500 setup file before you import the file into the SDS software Convert the 7500 results file before you import the file s into the qPCR STR system software Figure 1 on page 5 illustrates the files imported and exported throughout the automated HID workflow The Quantifiler Duo scripts Converter software and converted files are flagged with Download and install 1 Goto www tecan com HID software and scripts Click Download on the right side of the page under Product Details 3 Download the Quantifiler Duo scripts and Converter software 4 Unzip the downloaded files then follow the instructions included with the download to install the scripts and Converter Note The scripts must be installed on the qPCR STR system computer The Converter software is stand alone software not part of the Freedom EVOware or SDS software For ease of use we recommend that you install the Converter software on the qPCR STR system computer N Note The downloaded files also include instructions for using the Converter HID EVOlution qgPCR STR Setup System HID EVOlution Combination System Quantifiler Duo Converter Manual Tecan Document ID 396042 4 Quantifiler9 Duo DNA Quantification Kit User Bulletin Section 1 Usi
22. eating the detectors D IMPORTANT The detectors must have the exact names and capitalization shown below Detector Name Reporter Quencher Dye Dye Duo Human VIC Quencher Dye none make sure none is selected Duo Male FAM Quencher Dye none make sure none is selected Duo IPC NED Quencher Dye none make sure none is selected Y Note These detectors allow you to import the converted qPCR STR system generated 7500 setup file to the SDS Software Quantifiler Duo DNA Quantification Kit User Bulletin 5 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Prepare qPCR reagents and labware Prepare qPCR reagents and labware ENS Note Perform routine maintenance before placing reagents labware and samples on the worktable Refer to Chapter 2 Pre Run Procedures in the HID EVOlution qPCR STR Setup System Getting Started Guide Determine required reagent volumes e Note Applied Biosystems recommends that you combine reagents from different tubes from the same lot if necessary to meet the minimum volume requirements 1 Determine the volume of DNA standard and dilution buffer or volume of pre prepared DNA standard dilution series that you need You can either Letthe qPCR STR system prepare the DNA standard dilution series for you Use the table below to determine the required volume of undiluted Quantifiler Duo DNA Standar
23. erating Manual You have referred to the manufacturer s instrument documentation for important safety information related to the use of the Tecan Freedom EVO instrument SECTION 1 Section 1 Using the Quantifiler Duo DNA Quantification Kit with the HID EVOlution qPCR STR Setup System This section covers Workflow for the Quantifiler Duo DNA Quantification Kit usus 3 One time tasks 39 eer Re DER IRURE PESE MS 4 Download and install Quantifiler Duo scripts and Converter software 4 Create detectors for the 7500 Real Time PCR System 0 00 ee ee 5 Prepare qPCR reagents and labware ssssseeeeeeeeeeee 6 Determine required reagent volumes esses 6 Place reagents in the qPCR reagent block 0 cee eee 8 Set up the samples reagents and plasticware on the worktable 10 Run automated qPCR reaction setup 0 eee eee 11 Perform qPCR amplification and review results 0 0 cece eee eee 11 Convert and transfer the 7500 setup file lsseseeeeeeeeeee 11 Import the converted 7500 setup file and perform qPCR 06 12 Analyze and review the qPCR results 0 eens 13 Export and convert the 7500 results files 6 0 0 6 cece cece eee eee 14 STR PCR reaction set p izcsleles se ud eoe e ene Pee bande 15 Import the converted 7500 results files 20 eee eee 15 Quantifiler9 Duo DNA Quantification Kit User Bulletin
24. ftware v1 2 3 and the HID EVOlution system normalization and STR setup and that all sample information is complete and accurate EVOware software v1 4 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software 17 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Materials and methods Materials and methods Materials Component Description Converter HID EVOlution Quantifiler Duo Converter v1 0 0 0 qPCR STR system e Freedom EVO 150 robotic workstation e 4 channel LiHa e Freedom EVOware software see Experiments and results Freedom EVOware Software v1 4 on page 21 or Experiments and results Freedom EVOware9 Software v2 1 on page 28 for studies performed on software versions v1 4 SP1 Tecan HID EVOlution driver 1 0 0 24 and Tecan Sample Oriented EVOware Limited Edition SP1 or v2 1 Tecan HID EVOlution driver 1 0 0 25 SP4 Tecan Sample Oriented EVOware 1 2 SP1 Windows XP Professional operating system New qPCR setup scripts QuantifilerDuo plate QuantifilerDuo tubes Instruments and software e 3130xl Genetic Analyzer Data Collection Software v3 0 e GeneMapper D X Software version 1 0 e GeneAmp PCR System 9700 Thermal Cycler gold plated silver 96 well block e 7500 Real Time PCR System with SDS Software v 1 2 3 Consumables e 50 uL and 200 pL tips Tecan Group Ltd e TE buffe
25. g the MiniFiler PCR Amplification Kit or the Yfiler PCR Amplification Kit STR profiles were evaluated in the GeneMapper ID X software Results The results of contamination study are summarized in Table 11 One TE buffer blank exhibited Cy value 40 and was manually set up for amplification using the MiniFiler PCR Amplification Kit However this TE buffer blank did not exhibit an STR profile when amplified using the MiniFiler kit Therefore the lower Cy value is attributed to nonspecific amplification Table 11 TE buffer blank Cz and STR results N A samples not set up for amplification STR Results Sais RUE of Samples HTE HTEBlanksExhibiting tom Samp with C lt 40 MiniFiler Blank C1 40 MiniFiler Kit Kit Plate 44 None N A N A 36 None N A Tube 42 None N A N A 38 TEBlank24 position 47 No STR carrier 3 Duo Human Ct profile is 38 84 32 Quantifiler Duo DNA Quantification Kit User Bulletin Section 2 Validation of Quantifiler Duo Kit on HID EVOlution qPCR STR Setup System Conclusions Conclusions The Quantifiler Duo scripts and Quantifiler Duo converter were validated on the HID EVOlution qPCR STR Setup System using the Quantifiler Duo DNA Quantification Kit Precision and reproducibility studies confirm that the new Quantifiler Duo scripts and Quantifiler Duo converter provide reliable results at different DNA input amounts on Freedom EVOware Software v1 4 and
26. indication carrier 2 Duo Human profile 29 in carrier 2 profile of mixture Cy 36 85 exhibited Cy was STR value of 37 97 for detected profile male target with Yfiler Verification of normalization setup Experiment Normalization values The 7500 results files generated from Plate runs 1 2 and 3 and Tubes runs 1 2 and 3 from each system were processed with the Quantifiler Duo converter to generate human and male 7500 results files The converted human and male results files from each system were imported into the appropriate Quantifiler Duo script The dilution schemes shown in the script Sample Normalization Adjustment window that the scripts generated for each sample were evaluated The dilution schemes shown in the script Sample Normalization Adjustment window that the scripts generated for each sample were evaluated Total dilution T Dil values for each concentration of human and male DNA were examined to ensure that the calculated dilutions were appropriate for each concentration Refer to the HID EVOlution qPCR STR Setup System Getting Started Guide for information on the dilution protocols used by the qPCR STR system software Quantifiler9 Duo DNA Quantification Kit User Bulletin 27 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Experiments and results Freedom EVOware Software v2 1 Experiments and results Freedom EVOware Sof
27. n Kit STR profiles were evaluated in the GeneMapper ID X software The 7500 results files generated from Plate runs 1 2 and 3 and Tubes runs 1 2 and 3 from each system were processed with the Quantifiler Duo Converter to generate human and male 7500 results files The converted human and male results files from each system were imported into the appropriate Quantifiler Duo script The dilution schemes shown in the script Sample Normalization Adjustment window that the scripts generated for each sample were evaluated Experiments and results Freedom EVOware Software v1 4 Precision and reproducibility studies Experiment Slope and R On three consecutive days the two qPCR reaction plates generated from each qPCR setup run Figure 4 on page 20 were run on the 7500 Real Time PCR system six runs total Standard curve slope R2 and sample DNA concentration values were evaluated for the Ribonuclease P RNA Component H1 target RPPHI total human and Sex determining Region Y target SRY male Observed slope and R values are summarized in Table 5 The slope and R values were within the typical range of slope values observed during the development and validation of the Quantifiler Duo kit slope 3 0 to 3 6 and R 20 98 respectively Table 5 Slope and R values for standard curves Run Source plate Source tubes Duo Human Duo Male Duo Human Duo Male Slope R Slope R Slope R2 Slope R 1 3 02 0 997
28. ng the Quantifiler Duo DNA Quantification Kit with the HID EVOlution qPCR STR Setup System One time tasks Figure 1 Automated HID workflow for the Quantifiler9 Duo kit The Quantifiler Duo scripts Converter software and converted files are shown in red text and marked with Sample Information Instrument Software Sample Information Import Files Export Files Quantifiler Duo Converter Software qPCR STR qPCR reaction setup on the sample file qPCR STR system 7500 setup file barcode or with Quantifiler Duo scripts manual entry Converted qPCR on 7500 setup file 7500 Real Time PCR System with SDS Software v1 2 3 qPCR STR sample file S barcode or Normalization and manual ent STR PCR reaction setup on the qPCR STR system Quantifiler Duo Converter Software 7500 results file s CE setup file Converted human OR male 7500 STR PCR amplification on results file s 9700 Thermal Cycler CE on the 3130x CE setup file Genetic Analyzer with DC Software v3 0 CE results files Data Analysis on the CE results files GeneMapper D X Software Sample profiles STR results v1 0 Create detectors for the 7500 Real Time PCR System If you have not already done so create the following detectors in the SDS Software v1 2 3 for running the Quantifiler Duo kit assays Refer to the Applied Biosystems Quantifiler Duo DNA Quantification Kit User s Manual for details on cr
29. o DNA Quantification Kit Support and Validation New Scripts and Converter Software STR PCR reaction setup 16 Section 2 Validation of Quantifiler Duo Kit on HID EVOlution qPCR STR Setup System This section covers OVerview ge CR d REO TAE UO ur dan ud rit ers Materials and methods soir estreme cece ccc tee n Experiments and results Freedom EVOware Software v1 4 suus Experiments and results Freedom EVOware Software v2 1 0 0005 Conclusions sid duebebSengesheteuSseP bp Redux quus Quantifiler9 Duo DNA Quantification Kit User Bulletin SECTION 2 Overview The Quantifiler Duo DNA Quantification Kit Quantifiler Duo kit is now supported on the HID EVOlution qPCR STR Setup System qPCR STR system This section describes the validation and verification studies of quantitative PCR qPCR setup on the qPCR STR system using the new Quantifiler Duo scripts and the Quantifiler Duo Converter Software v1 0 that support the Quantifiler Duo kit The following studies were performed Precision and reproducibility studies to evaluate the ability to reproducibly set up Quantifiler Duo reactions using the Quantifiler Duo scripts EVOware9 software v1 4 and v2 1 Contamination studies to evaluate the potential for cross contamination EVOware software v1 4 and v2 1 Verification studies to evaluate that all files converted using the Quantifiler Duo Converter are compatible with the 7500 So
30. prepared using QuantiflerDuo plate script from DNA source plate Duo Male plate v2 1 Log10 of Quantity ng yL 0 01 0 025 01 as 1 2 5 10 50 DNA Concentration ng pL Quantifiler Duo DNA Quantification Kit User Bulletin 29 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Experiments and results Freedom EVOware Software v2 1 Figure 11 EVOware software v2 1 Quantification results obtained from the RPPH target total human in the qPCR reaction plates prepared using QuantiflerDuo tubes script from DNA source tubes Duo Human tubes v2 1 100 10 Log10 of Quantity ng pL 0 025 0 1 0 5 1 2 5 10 50 DNA Concentration ng pL Figure 12 EVOware software v2 1 Quantification results obtained from the SRY target male in the qPCR reaction plates prepared using QuantiflerDuo tubes script from DNA source tubes Duo Male tubes v2 1 100 10 0 1 Log10 of Quantity ng pL 0 01 0 025 0 1 0 5 1 2 5 10 50 DNA Concentration ng pL 30 Quantifiler9 Duo DNA Quantification Kit User Bulletin Section 2 Validation of Quantifiler Duo Kit on HID EVOlution qPCR STR Setup System Experiments and results Freedom EVOware Software v2 1 Table 9 RPPH1 target human DNA standard deviations and concentrations Male Sample Standard Deviations Expected ng n for Plate n Tubes
31. r Teknova Hollister CA Chemistry Kits Quantifiler Duo DNA Quantification Kit PN 4387746 Lot Number 0905008 AmpF STR Identifiler PCR Amplification Kit PN 4322288 Lot Number 0809108 AmpF STR Yfiler PCR Amplification Kit PN 4359513 Lot Number 0904046 AmpF STR MiniFiler PCR Amplification Kit PN 4373872 Lot Number 0904019 Sample DNA 1 female sample anonymous donor Serological Research Institute 223 L N 0909240 1 male sample Biochain Hayward CA 18 Quantifiler9 Duo DNA Quantification Kit User Bulletin Section 2 Validation of Quantifiler Duo Kit on HID EVOlution qPCR STR Setup System Materials and methods Extracted DNA sample plate and tubes set up The DNA concentrations in one anonymous female Serological Research Institute and one commercial male genomic DNA sample Biochain Institute were determined using the Quantifiler Duo DNA Quantification Kit by manual setup and in triplicate The samples were then serially diluted manually to obtain concentrations of 50 10 5 2 1 0 5 0 1 and 0 025 ng uL One sample plate containing extracted DNA source plate and one set of 1 5 mL tubes containing extracted DNA source tubes were prepared manually with 2 to 4 replicates for each concentration and TE buffer blanks Figures 2 and 3 Figure 2 Sample plate set up with extracted DNA source plate 1 2 3 4 5 6 7 8 9 10 11 A BU ngiuL TE Blank 8 ng uL
32. re within the typical range refer to the Quantifiler Duo DNA Quantification Kit User s Manual If necessary omit standard outliers then click Analyze to recalculate the standard curve 3 Review the quantitation data and make the necessary determinations a Review the quantitation data including the IPC Cy and the ratio of male to female DNA and consider the original condition of the sample b For each sample determine if Thesample requires additional processing before STR PCR reaction setup You will proceed with autosomal STR or Y STR reaction setup D IMPORTANT The qPCR STR system sets up STR PCR reactions with one AmpF STR kit per run We recommend that you view your qPCR results in the SDS software then determine how you will batch samples for amplification with the appropriate AmpF STR kit You need to adjust the target input amount to add more or less DNA to the amplification reaction hy Note After you import the qPCR results to the qPCR STR system software you can adjust the target input amount in the Sample Normalization Adjustment window Note Refer to Interpretation of Results in the Quan tifiler Duo DNA ES Quantification Kit User s Guide for details Quantifiler9 Duo DNA Quantification Kit User Bulletin 13 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Perform qPCR amplification and review results Export and convert the 7500 re
33. sults files W Note During STR PCR reaction setup you import the converted 7500 results gt files to the qPCR STR system The EVOware uses the converted 7500 results file to normalize the DNA input amount for STR PCR reaction setup on the qPCR STR system When you are done analyzing the results in the SDS software and have determined the appropriate AmpF STR kit for use with each sample export the 7500 results file s convert the file s then copy them to the qPCR STR system computer 1 In the SDS software select File Export Results 2 In Save as type select Results Export Files csv then click Save 3 Use the Converter to create two results files a human file and a male file e Note Refer to the HID EVOlution qPCR STR Setup System HID EVOlution Combination System Quantifiler amp Duo Converter Manual Tecan Document ID 396042 for details 4 Copy the converted results files to the computer where the Freedom EVOware software is installed or to a server that can be accessed from both systems 14 Quantifiler9 Duo DNA Quantification Kit User Bulletin SECTION 1 STR PCR reaction setup When performing STR PCR reaction setup using samples quantified with the Quantifiler Duo kit 1 Prepare STR PCR reagents and labware as described in Chapter 6 of the HID EVOlution qPCR STR Setup System Getting Started Guide 2 Run STR PCR setup as described in Chapter 7 of the HID EVOlution
34. the Duo Male target and was manually set up for amplification using the Yfiler and MiniFiler PCR Amplification kits However this sample did not yield a Y STR profile when amplified using the Yfiler PCR Amplification Kit Similarly the autosomal STR profile obtained using the MiniFiler kit did not indicate the presence of a mixture Therefore the lower Cy values are attributed to nonspecific amplification Quantifiler9 Duo DNA Quantification Kit User Bulletin Section 2 Validation of Quantifiler Duo Kit on HID EVOlution qPCR STR Setup System Experiments and results Freedom EVOware Software v1 4 Table 8 TE buffer blank Cz and STR results N A samples not set up for amplification STR Female STR Results Total TE Blanks Exhibiting Results Total samples with Run So igs Ceki tor male TE C1 40 for male and or P T 4 MiniFiler blanks human detector MiniFiler detector Yfiler Kit Kit Kit Plate 1 44 None N A N A 36 None N A Plate 2 44 None N A N A 36 TEBlank 5 well A2 Duo No STR Human Cy 37 03 profile Plate 3 44 None N A N A 36 TEBlank 17 well B5 No STR Duo Male C12 37 65 profile TEBlank 20 well H5 No STR Duo Human C7 39 20 profile Tube 1 42 None N A N A 38 None N A Tube 2 42 None N A N A 38 TEBlank 3 position 6 No STR carrier 1 Duo Male Cr profile 38 20 Tube 3 42 2 ng L female No Male No 38 TEBlank 15 position 30 No STR sample position STR
35. tification Kit User s Guide for details 3 Import the converted 7500 setup file into the Applied Biosystems 7500 SDS Software plate document for use during the quantitation run as follows a In the SDS software select File New to create a new Absolute Quantification Standard Curve plate document then in the new document wizard select Finish b With a blank plate document open select File Import Sample Setup C Browse to locate the converted 7500 setup file select the file then click Open d In the plate document select the Instrument tab then Change the Sample Volume to 25 uL Select the 9600 Emulation box Note The thermal cycler protocol validated for use with the EI Quantifiler Duo kit includes a hold step at 50 C for 2 minutes e Omit any blank wells 12 Quantifiler9 Duo DNA Quantification Kit User Bulletin Section 1 Using the Quantifiler Duo DNA Quantification Kit with the HID EVOlution qPCR STR Setup System Perform qPCR amplification and review results 4 Save the plate document a Select File Save As b In the Save as dialog box Enter a file name in the File name field Do not add an extension to the file name Select SDS Document sds from the Save as type drop down list Click Save 5 Select the Instrument tab then click Start Analyze and review the qPCR results 1 In the SDS software click Analyze 2 Review the standard curve to ensure that results a
36. tifiler Duo Dilution Buffer tube i The required number of tubes with the Quantifiler Duo Primer Mix depends on the number of reactions and the fill level of the tubes The instrument aspirates 190 uL at a time In order for the instrument to detect reagent in a tube the tube must contain a minimum of 240 uL 190 pL plus a 50 pL dead volume If the volume of reagent in the first tube position 9 falls below 240 pL the instrument looks for a sufficient volume in the position 10 tube then in the position 11 tube If the tubes are not available or the instrument does not detect sufficient volume the instrument pauses the run and displays the error message Not enough liquid Quantifiler9 Duo DNA Quantification Kit User Bulletin 9 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Prepare qPCR reagents and labware Set up the samples reagents and plasticware on the worktable Set up the samples reagents and plasticware as described in Chapter 3 of the HID EVOlution qPCR STR Setup System Getting Started Guide and shown below in Table 4 Note The TE buffer trough 7 in Table 4 below is not used for Quantifiler Duo kit runs Dilution buffer is provided with the kit and is placed in the reagent block Table 4 Tecan Freedom EVO workstation worktable layout for a qPCR setup run loocooocoo CRN IX UD e OOo Y Y 00000000 00000000 Site number 1
37. tware v2 1 Precision and reproducibility study Experiment The source plate Figure 2 on page 19 and source tubes Figure 3 on page 19 were run using the QuantifilerDuo plate script The two qPCR reaction plates were run on the 7500 Real Time PCR system Sample DNA concentration were evaluated DNA concentration The range of variation in the quantification results is Comparable to the results seen during developmental validation reported in the Quantifiler Duo PCR Amplification User Guide at all tested concentrations of DNA e Similar for source plate and source tubes DNA concentrations are summarized in Figures 9 through12 on pages 29 and page 30 The standard deviation values and concentration ranges for human and male DNA quantity values are summarized in Tables 9 and 10 on page 31 28 Quantifiler9 Duo DNA Quantification Kit User Bulletin Section 2 Validation of Quantifiler Duo Kit on HID EVOlution qPCR STR Setup System Experiments and results Freedom EVOware Software v2 1 Figure 9 EVOware software v2 1 Quantification results obtained from the RPPH1 target total human qPCR reaction plates prepared using QuantiflerDuo plate script from DNA source plate Duo Human plate v2 1 100 10 Log10 of Quantity ng pL 0 1 0 01 0 025 01 0 5 1 2 5 10 50 DNA Concentration ng pL Figure 10 EVOware software v2 1 Quantification results obtained from the SRY target male in the qPCR reaction plates
38. uL Plate for Tubes 0 025 4 0 008 4 0 007 0 1 4 0 024 2t N At 0 5 4 0 080 4 0 054 1 4 0 161 4 0 183 2 4 0 354 4 0 120 5 4 0 450 4 0 324 10 4 0 992 4 0 319 50 4 7 082 4 5 746 Female Concentration ng uL Sample Expected ng n for Plate n for Tubes Tubes pL Plate 0 5 2 0 59 0 71 2 0 61 0 54 1 2 1 29 1 35 2 1 10 1 18 2 2 2 52 259 2 2 05 1 93 5 2 7 73 7 27 2 6 12 5 17 10 2 14 70 14 84 2 12 52 12 52 50 2 90 35 83 46 2 79 98 75 85 f Due to the number of wells on the qPCR reaction plate only two samples were run Standard deviation not calculated Table 10 SRY target male DNA standard deviations and concentrations Male Standard Deviations Sample Expected n Plate n Tubes ng pL for for Tubes Plate 0 025 4 0 015 4 0 011 0 1 4 0 044 2 N A 0 5 4 0 053 4 0 056 1 4 0 243 4 0 179 2 4 0 512 4 0 130 5 4 0 645 4 0 518 10 4 1 223 4 0 928 50 4 3 328 4 2 727 Quantifiler Duo DNA Quantification Kit User Bulletin 31 Quantifiler Duo DNA Quantification Kit Support and Validation New Scripts and Converter Software Experiments and results Freedom EVOware Software v2 1 Contamination study Experiment Cy values from the precision and reproducibility studies were evaluated If any female samples exhibited Cy values 40 for the Quantifiler Duo Male target and if any TE buffer blanks exhibited Cy values 40 they were manually set up for amplification usin
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