Manual
Contents
1. Thaw all reagents and keep on ice Spin down the reagents before use A blank control water and a positive control your sample control is suggested to run with samples each time A 1 A 2 A 3 A 4 A 5 A 6 A 7 A 8 Collect and label PCR tubes or a 96 well PCR plate as follows Sample 1 2 3 DAO OOOO Neg Negative control water Pos Positive control CTL WR Add 26u of Master mix into each tube Add 1ul of PCR primer mix PCR P to each tube Add 3ul of nuclease free water to the Neg tube for negative control Add 3ul of CTL WR to the Pos tube for positive control Add 3ul of sample DNA 20 80 ng ul to correspondent tube Cap the tubes and place the tubes to thermal cycler Run Program 1 Program 1 1 cycle 95 C 5 min 35 cycles 94 C 30 sec 52 C 45 sec 72 C 45 sec 1 cycle 72 C 5 min Hold at 4 C Technical Support 1 888 825 6005 11 Y UOISJ A TrimGen Mutector II Warfarin Genotyping Note y Option To verify the PCR product load 5 ul of the PCR product onto 1 2 agarose gel and use 100 bp size markers to confirm the PCR products The three PCR product sizes are VKORC1 167 bp CYP 2C9 2 174 bp CYP2C9 3 221 bp V The procedure can be temporarily stopped after Program 1 Store the PCR product in 4 C for next day testing PCR Product Clean up B 1 Collect new 0 2 ml tubes One tube for each PCR reaction Label the tubes the same2. UOISJ A Discard the Collection Tubes and transfer the TF Filters into C 2 Add 11 1 of ST WR into all tubes a new Collection Tube Label the Collection Tubes with sample ID The TF Filters are ready for use C 3 Add 2u of DP WR into all tubes D 5 After the ST reaction load all ST reaction contents 15 C 4 Add 2ul of E1 treated Negative PCR control to the Neg onto the top of the gel in each pre prepared TF Filter tube et D 6 Centrifuge the TF Filters at 1 000 x g 2 000 3000 rpm for C 5 Add 2ul of E1 treated Pos to the Pos tube most tabletop centrifuge for 2 3 minutes C 6 Add 2ul of E1 treated Sample to each corresponding sample tube D 7 Discard the TF Filters The solution in the tubes contains ST product and is ready for sample loading C 7 Cap the tubes mix the contents and spin all tubes C 8 Place the tubes into thermal cycler and perform ST reaction E Sample Loading using Program 3 g rogram E 1 Add 15yl of Loading buffer to each well of a sequencer adapter plate Program 3 E 2 Transfer 2 4ul of the filtered ST products into each well 1 cycle 94 C 4 min H p 20 cycles 94 C 30 sec GE Do not mix sample by pipetting the mixture up down It may 50 C 45 sec Na generate bubbles Signal may vary depending on the 70 C 30sec instrument used It is recommended to adjust the loading Hold at 4 C volume 2 4ul to optimize the signal on your machine If the signal is too strong
3. as the PCR tubes B 2 Add 11 ul of E1 Mix to each new tube B 3 Transfer 4ul of PCR product to each tube The remaining PCR products can be stored at 20 C for re testing f PCR product may cause lab contamination To avoid the PCR product contamination use the filter tip for the pipetting handle with care and perform this step in a designated area After transfer clean up the work area and change gloves B 4 Cap the tubes mix the contents and spin all tubes B 5 Incubate the tubes in thermal cycler using Program 2 Program 2 37 C for 30 min 95 C for 5 min Hold at 4 C Note V The procedure can be temporarily stopped after Program 2 Store the reaction tube in 4 C for next day testing Technical Support 1 888 825 6005 12 Y UOISJ A TrimGen Mutector II Warfarin Genotyping TrimGen Mutector II Warfarin Genotyping c ST Reaction D ST Product Clean up C 1 Collect 0 2ml strip tubes 1 tube per sample and 2 control tubes for each test run Label the tubes as follows Filter preparation D 1 Collect the TF Filters and Collection Tubes one set for each ST reaction E1 treated PCR controls D 2 Snap off the bottom portion of the filter tip ref page 7 for Sc Zaoa snap off line Centrifuge the TF Filters at 1 000 x g 2 000 3000 rpm for most tabletop centrifuge for 2 3 minutes to remove the E1 treated PCR Samples 1 2 3 excess buffer from the filters Y
4. dilute the ST product with water 3 5 v The procedure can be temporarily stopped after Program 3 times and re loading the sample Store the reaction tube at 4 C for next day testing E 3 Load the plate to sequencer and run the pre set Data Collection Program ref page 8 Technical Support 1 888 825 6005 Technical Support 1 888 825 6005 13 14 Y UOISJ A TrimGen F Data Analysis Mutector II Warfarin Genotyping The CTL WR Genotype controls shows all genotypes color and size Use these controls as a standard to identify peak s present in the samples Results for mutation controls Red VKORC1 209 2 1639 A 430 T Red 2C9 2 430C Blue AJ Oe ne DO A Peak Color Peak Size Interpretation 1 Blue 20 27 VKORC1 1639 G 2 Red 23 54 VKORC1 1639 A 3 Blue 43 09 2C9 2 430C 4 Red 45 75 2C9 2 430T 5 Blue 53 86 2C9 3 1075C 6 Red 57 96 2C9 3 1075A The data were produced using POP7 and 36 cm capillary The peak size may vary slightly depending on instrument polymer type and the length of capillary Customer can confirm the correct peak size using the CTL WR Technical Support 1 888 825 6005 15 Y UOISJ A TrimGen Notes Mutector II Warfarin Genotyping Technical Support 1 888 825 6005 16 Y UOISJ A
5. single nucleotide variation through multiple steps 1 Sequence specific amplification of target gene 2 Sequence selective termination of target nucleotide and 3 Sequence dependent primer extension Y UOISJ A Enzymatic cleanup of PCR product The genotypes are easily differentiated by fragment size and colors to give 40 min clear cut results ST reaction Shifted Termination Assay STA Shifted Termination Assay is a proprietary technology that uses a combination of uniquely designed primers mixtures of modified enzymes and specially synthesized nucleotides The STA reaction recognizes wild type or mutant target sequences and selectively terminates or extends the detection primers with 1 to 20 nucleotides This extension is repeated 20 times with labeled nucleotides to enrich the detection signal The enriched signals are then easily detected by fragment analysis 1 hour Cleanup of ST product 10 mi Wild type i 0O Wild type gt X Mutant Mutant 0 2 N X Fragment analysis 40 min Capillary sequencer Technical Support 1 888 825 6005 Technical Support 1 888 825 6005 5 6 Y UOISJ A TrimGen Mutector II Warfarin Genotyping Materials Provided The Mutector II Warfarin Kit contains reagents for 32 tests Materials Cap label Quantity PCR Primer Mix PCR P 50 pl Master Mix MM 1000 ul E1 Mix E1 430 ul ST WR ST 430 ul DP WR DP 80 pl CTL WR CTL 50 ul Loa
6. Mutector II Warfarin Genotyping kit Cat No GP03 User Manual V1 4 Storage Upon receipt of the kit store at 20 C until use At this temperature the reagents are stable for 1 year After first use store all of the reagents at 2 8 C and keep them protected from direct light At this condition the reagents are stable for 3 months For research use only not for use in diagnostic procedures TrimGen Mutector II Warfarin Genotyping Technical Support 1 888 825 6005 2 Y UOISJ A TrimGen Mutector II Warfarin Genotyping CONTENTS Introduction Overview of Mutector II Detection Materials Provided Materials Required Equipment Required Sample Preparation Sequencer setup Thermal Cycling Programs Mutector II Assay Protocol PCR Amplification PCR Product Clean up ST Reaction ST Product Clean up Sample Loading Data Analysis Technical Support 1 888 825 6005 3 pL UOISJ AN TrimGen Limited Product Warranty It is imperative that the users strictly adhere to this manual Failure to do so will void TrimGen s guarantee of this product TrimGen Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose Notice to Purchaser The Mutector II kit is provided as a research use only product The purchaser must determine the suitability of the product for their particular use No cl
7. aim or representation is intended for use of this product to identify any specific organism or for a specific clinical use diagnostic prognostic therapeutic or blood banking The purchase of Mutector II kit includes a limited nonexclusive license to use the kit This license does not grant rights to reproduce or modify the Mutector I kit for resale or to use the Mutector II kit to manufacture commercial products without written approval of TrimGen Corporation No other license expressed implied or by estoppels is granted Product Safety and Liabilities When working with the kit reagents always wear a lab coat disposable gloves and protective goggles TrimGen Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the misuse the results of use or the inability to use this product Technical Support 1 888 825 6005 4 Mutector II Warfarin Genotyping Y UOISJ A TrimGen Mutector II Warfarin Genotyping TrimGen Mutector II Warfarin Genotyping Introduction Overview of Mutector II Detection The Mutector I1 Warfarin genotyping assay is a single tube test designed for identifying the following single nucleotide polymorphisms SNPs CYP2C9 2 C430T CYP2C9 3 A1075C VKORC1 1639 G gt A PCR amplification 1 5 2 hours The assay uses TrimGen s proprietary technology called Shifted Termination Assay STA The STA technology accurately detects
8. com docs Partll Data Collection Setup pdf Step Ill Data Analysis Using GeneMapper www trimgen com docs Partlll Data Analysis GeneMapper pdf GeneScan Analysis Step I Data Collection Software Setup www trimgen com docs Partll Data Collection Setup pdf Step Il GeneScan Setup and Data Analysis www trimgen com docs PartlV Genescan pdf Technical Support 1 888 825 6005 9 Y UOISJ A TrimGen Mutector II Warfarin Genotyping Gi Important Spectral calibration is required before running the test To read the test results correctly the sequencer needs to be calibrated with the DS 32 calibration kit Applied Biosystems Cat No 4345831 This is a one time calibration to set up correct spectral channels to read the test results Refer to the DS 32 Matrix standards kit to prepare the DS 32 matrix standards Run a Matrix Standard Set DS 32 5FAM JOE NED ROX to perform a spectral calibration Thermal Cycling Programs Program 1 PCR 1 cycle 95 C 5 min 35 cycles 94 C 30 sec 52 C 45 sec 72 C 45 sec 1 cycle 72 C 5 min Hold at 4 C Program 2 E1 treatment 37 C 30 min 95 C 5 min Hold at 4 C Program 3 ST reaction 1 cycle 94 C 4 minute 20 cycles 94 C 30 sec 50 C 45 sec 70 C 30 sec Hold at 4 C Technical Support 1 888 825 6005 10 Y UOISJ AN TrimGen Mutector II Warfarin Genotyping Mutector II Assay Protocol A PCR Amplification
9. ding Buffer LB 1200 ul TF Filters N A 32 Collection Tubes N A 32 Light sensitive Keep these reagents protected from direct light Reagents Description PCR Primer Mix PCR primer mix for amplification of VKORC1 and CYP2C9 gene Master Mix Pre mixed reagents for PCR amplification E1 Mix Enzyme mix for cleanup of PCR products ST WR Light sensitive Pre mixed reagents for signal enrichment and detection DP WR Pre mixed detection primers CTL WR Pre mixed genotype control DNA Loading Buffer Light sensitive Contains the loading buffer for ABI capillary type sequencers and special fluorescent labeled size standards Technical Support 1 888 825 6005 7 pL UOISJ A TrimGen Mutector II Warfarin Genotyping TF 50 Filter TF 50 filter is a tip filter designed to remove free fluorescent dyes from the reaction mixture Dry Store the tip filter in 2 8 C If the buffer on top of the gel evaporates Dry see picture on left add 100 150ul deionized water to re hydrate Snap the gel If the gel is completely off line dried white in appearance it is necessary to soak the gel overnight after adding water Good Completely dried Before use snap off the tip at the position of Snap off line Materials required 0 2 ml PCR tubes 8 well strip tube DS 32 Matrix Standard Kit for the 3100 and 3130 Series Systems one time set up Applied Biosystems Cat No 4345831 Equipment r
10. equired Thermal Cycler Any type of thermal cycler with a 0 2 ml tube block is acceptable for performing the Mutector II assay Sequencer Applied Biosystems capillary type Genetic DNA Analyzer Analysis Software Data Collection software for ABI capillary sequencer GeneMapper for fragment analysis or GeneScan Technical Support 1 888 825 6005 8 Y UOISJ A TrimGen Mutector II Warfarin Genotyping DNA Sample Preparation Reagents for DNA preparation are not provided with the Mutector II kit Paraffin FFPE and fresh or frozen tissue samples A kit specially designed for FFPE sample DNA extraction is available at TrimGen WaxFree DNA Cat WF 50 for 50 samples WF 100 for 100 samples Blood Any commercially available DNA extraction kit is acceptable DNA concentration When using commercial DNA extraction kit for DNA extraction adjust the final concentration of extracted DNA to 20 80 ng ul When using TrimGen s DNA preparation kit follow the kit protocol to perform the PCR reaction Sequencer setup First time users should set up the analysis program for the ABI sequencer one time setup After setup the program can apply to all Mutector II tests for data analysis Please choose either GeneMapper or GeneScan to analyze your data GeneMapper Analysis Step I GeneMapper Setup www trimgen com docs Partl GeneMapper Setup pdf Step Il Data Collection Software Setup www trimgen
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