User Manual V1.6
Contents
1. 2 x 1 1 Total number of sample DP BF 2 x 2 x 1 1 Total number of sample For negative and positive controls For pipetting error Transfer entire volume of the reagents to one tube and mix the contents This is the ST mix C 2 Collect 0 2ml strip tubes one for each PCR reaction Label the tubes with sample ID Label the tubes as follows Controls 86000 Samples 1 2 3 C 3 Add 13 uL of ST Mix from step C 1 into each of the tubes C 4 Add 2 uL of C UP treated Negative PCR control to the Neg tube C 5 Add 2 uL of C UP treated Pos to the Pos tube C 6 Add 2 uL of C UP treated Sample to each corresponding sample tube C 7 Mix the contents and spin all tubes 12 BRAF V600 mutation detection kit GP02 EkK User Manual V1 6 C 8 Place the tubes into the thermal cycler and perform the ST reaction using Program 3 Program 3 1 cycle 94 C 4 min 20 cycles 94 C 20 sec 55 C 30 sec 72 C 20 sec Hold at 4 C D ST Product Clean up Filter preparation D 1 D 2 D 3 D 5 D 6 D 7 Collect the TF Filters and Collection Tubes one set for each ST reaction Snap off the bottom portion of the filter tip ref page 7 for snap off line Centrifuge the TF Filters at 1 000 x g 3000 rpm for most tabletop centrifuge for 2 3 minutes to remove the excess liquid from the filters Discard the Collection Tubes and move the TF Fil2. the 2 marker and 80 the 6 marker along the X axis see figure 1 Find the results for the mutation controls CTL BF The mutation controls CTL BF will show a total of 5 peaks Use these controls as a standard to identify peak s present in the samples 35 45 V600E red V600A i blue Pj type a veooKr n V600G ging red 60 Sample analysis A wild type WT peak is presented in every sample and serves as an internal control Low peak height 14 BRAF V600 mutation detection kit GP02 Ek User Manual V1 6 indicates low concentration or poor quality of input DNA If the peak is not observed it indicates that the DNA amplification failed or the sample is 100 mutant such as mutant cell lines When a sample contains a mutation an additional peak shows up sometimes there are multiple peaks due to an existence of multiple mutations To identify the mutation always compare the peak size and color with the peaks of the Mutation Controls CTL BF Any peak that does not match with the mutant controls will not be considered The peak size may slightly shift due to migration differences between capillary tubes The size shift can be identified by comparing the wild type peak in the sample to the wild type peak in the Mutation Controls CTL BF When the shift occurs the peak size for the mutations will have a proportional shift G Troubleshooting G1 Color leak through When the
3. BRAF V600 mutation detection kit GP02 EkK User Manual V1 6 Mutector II BRAF V600 Mutation Detection Kit Cat No GP02 EK User Manual V1 6 For research use only not for use in diagnostic procedures BRAF V600 mutation detection kit GP02 EkK User Manual V1 6 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 research use only not for use in diagnostic procedures The purchaser must determine the suitability of the product for their particular use The purchase of the Mutector II kit includes a limited nonexclusive license to use the kit This license does not grant rights to reproduce or modify the Mutector II 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 u
4. ct spectral channels to read the test results Refer to the DS 32 Matrix standards kit to prepare the DS 32 matrix standards Runa Matrix Standard Set DS 32 5FAM JOE NED ROX to perform a spectral calibration Thermal Cycling Programs Program 1 PCR 1 cycle 94 C 5 min 35 cycles 94 C 30 sec 52 C 30 sec 72 C 30 sec 1 cycle 72 C 5 min Hold at 4 C Program 2 PCR product clean up 37 C 25 min 95 C 5 min Hold at 4 C Program 3 ST reaction 1 cycle 94 C 4 minute 20 cycles 94 C 20 sec 55 C 30 sec 72 C 20 sec Hold at 4 C BRAF V600 mutation detection kit GP02 EK User Manual V1 6 Mutector II Assay Protocol A PCR Amplification Thaw all reagents and keep on ice Spin down the reagents before use Mutation controls CTL BF are required for each test run A negative control water is suggested to run with the samples each time A 1 Collect 0 2 ml PCR strip tubes and label the tubes as follows Neg Negative control water Pos Positive control CTL BF A 2 Prepare PCR reaction mix Master Mix 18 x 2 x 1 1 Total number of sample PCR Primers 1x 2 x 1 1 Total number of sample For negative and positive controls For pipetting error Transfer entire volume of the reagents to one tube and mix the contents This is the PCR reaction mix A 3 Add 19 uL of PCR reaction mix into each tubes A 4 Add 1 uL of nuclease free wa
5. e The sample needs to be re loaded The ST products will compete with the size standard DNA to enter the capillary tube 15 BRAF V600 mutation detection kit GP02 EK User Manual V1 6 G4 G5 G6 G7 The high sample signal will reduce the size standard signal Diluting the final ST product with de ionized water will easily resolve this problem The size standard may be miscalculated Check the size standard and manually correct the size standard and reanalyze the data Peak size shift Sometimes the peak size may vary between the capillary tubes Air bubbles or capillary tube conditions could cause the shift Comparing the wild type peaks in the sample and Mutation Controls can identify the peak size shift If the shift occurs the mutation peaks will show a similar shift Extra peaks do not maich any peaks in CTL BF controls In some circumstances for example sample DNA is too low or PCR is not amplified properly the PCR primers may form primer dimers These primer dimers can generate blue peak s with different sizes Any peaks that do not match the CTL BF controls are not considered a mutation signal Background noise The background of the test is normally low and high background noise may be caused by poor quality DNA When the PCR reaction does not perform properly the intensity of the wild type peak is usually lower than 500 and many small peaks are observed These peaks are not considered as signal Mutat
6. ion peak cut off For some samples a small peak may be observed in one of the mutation positions To verify the peak you need to confirm the signal strength of the wild type peak If the wild type peak is too high can not see the top of the peak and the peak is highlighted with pink color your ST reaction is too strong and the small peak may be pull up from background noise Follow trouble shooting 2 to dilute the final product of the ST reaction with de ionized water After dilution reload the sample If you can see the top of the wild type peak use the following calculation to identify the small peak Ratio Area of mutant peak Area of wild type peak If the ratio is larger than 0 06 the peak is determined to be a mutation peak the ratio does not represent the percentage of the mutation in the sample Otherwise the peak is a background pull up and does not indicate the presence of a mutation in the sample 16
7. le from TrimGen Product information WaxFree DNA for 50 samples Cat WF 50 WaxFree DNA for 100 samples Cat WF 100 DNA concentration When using a column or bead DNA extraction method adjust the final concentration of extracted DNA to 20 80 ng pL 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 sequencer one time setup After setup the program can apply to all Mutector Il 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 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 paf BRAF V600 mutation detection kit GP02 EK User Manual V1 6 an 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 corre
8. sample DNA concentration is too high the ST reaction generates a strong fluorescent signal gt 5 000 rfu Fluorescence spillover will occur For example the black peak of the wild type signal may be observed in the red and or blue channels This color spillover is caused by limitation of the instrument The leak through peak will have the exact same peak size as the original peak Because the mutation peaks have different peak size leak through will not affect data analysis G2 The peak signal is too high The assay is set at a condition to detect mutations in a small sample such as DNA extracted from fine needle aspiration FNA sample For regular FFPE sample the assay signal may be too high to analyze peak height gt 8000 rfu can not see the top of the peak or the peak is highlighted with pink color Diluting the final STA product with de ionized water can efficiently reduce the signal and optimize the peak height Do not dilute the assay reagents it will cause improper enzymatic reaction and generate a miss call Each laboratory has different PCR instrument s the signal intensity may vary among the laboratories first time users should perform a titration of final STA product Once the dilution factor is determined the assay will show consistent results G3 Graphic data will not automatically show Check the raw data If the signals from the sample and size standards are too low the capillary tube may be blocked by a bubbl
9. se or the inability to use this product BRAF V600 mutation detection kit GP02 EK User Manual V1 6 CONTENTS Introduction Overview of Mutector II Assay Materials Provided Materials Required Equipment Required DNA Sample Preparation oOo N NO OO RA Sequencer Setup Thermal Cycling Programs 9 Mutector II Assay Protocol 10 Data Analysis 15 Troubleshooting 16 Storage Upon receipt of the kit store at 20 C until use At this temperature the reagents are stable for 8 months 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 2 months BRAF V600 mutation detection kit GP02 EK User Manual V1 6 Introduction The Mutector II BRAF V600 mutation detection kit GP02 EK uses TrimGen s proprietary technology called Shifted Termination Assay STA to detect mutations in codon V600 of the BRAF gene This single tube assay detects the following mutations V600E GTG gt GAG V600A GTG gt GCG V600G GTG gt GGG V600K GTG gt AAG V600R GTG gt AGG The STA technology enriches mutation signals and is able to detect low levels of somatic mutations Mutector II has sequencing like accuracy through multiple steps 1 PCR amplification 2 Selective sequence hybridization 3 Sequence dependent termination and specific primer extension by STA Shifted Termination Assay STA Shifted Termination Assay STA
10. t sensitive Keep these reagents protected from direct light Reagents Description Master Mix Reagents for DNA amplification PCR P PCR primer mix for amplification of BRAF gene C UP1 and C UP2 Enzyme mix for cleanup of PCR products C UP Buffer Buffer for the C UP enzymes ST BF Light sensitive Pre mixed reagents for mutation enrichment and detection DP BF Pre mixed detection primers BRAF V600 mutation detection kit GP02 EK User Manual V1 6 CTL BF Pre mixed mutant and wild type DNA The controls are sufficient for 16 test runs Loading Buffer Light sensitive Loading buffer for ABI capillary type sequencers and special fluorescence labeled size standards 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 required 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 BRAF V600 mutation detection kit GP02 EK User Manual V1 6 DNA Sample Preparation Any commercially available DNA extraction kit is acceptable Paraffin FFPE tissue samples A high efficiency DNA extraction kit for FFPE sample is availab
11. technology is a proprietary multi base primer extension method The specially formulated ST reagents extend detection primers by multiple bases to increase signal strength and fragment size creating mutation peaks that are easily distinguished from wild type The STA technology has sequencing like accuracy and the mutation is confirmed by both peak color and fragment size The method enriches mutation signal and detects low level mutations missed by sequencing Mutant O Wild type X Mutant Wild type a eX Fragment analysis STA reaction BRAF V600 mutation detection kit GP02 EK User Manual V1 6 Overview of Mutector II Assay Single tube assay for each sample PCR amplification 1 2 hours Time varies depending on the type of thermal cycler used C UP treatment PCR product clean up 30 min ST reaction Mutation enrichment and detection 45 min Time varies depending on the type of thermal cycler used Load to sequencer Wild type Capillary Electrophoresis Fragment analysis Mutation ev BRAF V600 mutation detection kit GP02 EK User Manual V1 6 Materials Provided The Mutector Il BRAF Mutation kit GPO2 EK contains reagents for 32 reactions Materials Quantity Master Mix 650 uL PCR P 50 pL C UP1 40 uL C UP2 40 uL C UP Buffer 350 uL ST BF 400 pL DP BF 80 uL CTL BF 50 uL Loading Buffer 650 uL TF Filters 32 Collection Tubes 32x 2 Ligh
12. ter to the Neg tube A 5 Add 1 uL of CTL BF to the Pos tube A 6 Add 1 uL of sample DNA 20 80 ng ul to each sample tube 10 BRAF V600 mutation detection kit GP02 EK User Manual V1 6 A 7 Place the PCR tubes in a thermal cycler and run Program 1 Program 1 1 cycle 94 C 5 min 35 cycles 94 C 30 sec 52 C 30 sec 72 C 30 sec 1 cycle 72 C 5 min Hold at 4 C The procedure can be temporarily stopped after Program 1 The PCR product can be stored at 4 C for next day test During the PCR amplification prepare steps B1 B2 B PCR Product Clean up Prepare the C UP mix C UP1 1 0 x number of samples x 1 1 uL C UP2 1 0 x number of samples x 1 1 uL C UP Buffer 10 x number of samples x 1 1 uL For pipetting error B 1 Collect 0 2ml strip tubes one tube for each PCR reaction Label the tubes the same as the PCR tubes B 2 Add 12 uL of C UP Mix to each new tube B 3 Transfer 5 uL of PCR products to each corresponding tube B 4 Cap the tube mix the contents and spin all tubes B 5 Incubate the tubes in a thermal cycler using Program 2 Program 2 37 C for 25 min 95 C for 5 min Hold at 4 C During the incubation prepare steps C1 C4 11 BRAF V600 mutation detection kit GP02 Ek User Manual V1 6 C ST Reaction Mutation enrichment C 1 Collect one 2ml tube and label the tube with ST Prepare ST Mix using formula below ST Mix ST BF 11 x
13. ters into a new Collection Tube Label the Collection Tubes with sample ID The TF Filters are ready for use After the ST reaction load all ST reaction contents 154l onto the top of the gel in each pre prepared TF Filter Centrifuge the TF Filters at 1 000 x g 3000 rpm for most tabletop centrifuge for 2 3 minutes Discard the TF Filters The solution in the tubes contains ST product and is ready for sample loading 13 BRAF V600 mutation detection kit GP02 EK User Manual V1 6 E E 1 E 2 E 3 F1 F2 F3 F4 F5 F6 Sample Loading Add 15 uL of the Loading buffer to each well of a sequencer adapter plate Transfer 2 4 uL of the filtered ST products into each well Signal may vary depending on the instrument used It is recommended to adjust the loading volume to optimize the signal on your machine If the signal is too strong dilute the ST product with water 3 5 times before loading the sample Load the plate to sequencer and run the pre set Data Collection Program ref page 8 Data Analysis Open the analysis software GeneMapper or GeneScan Follow the instructions to add the data for analysis The instructions are provided online GeneMapper www trimgen com docs Partlll Data Analysis GeneMapper pdf GeneScan www trimgen com docs PartlV Genescan pdf In the sample plot window shows graphic data zoom in the graphic by selecting area between size markers 25
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