User Manual V1.1
Contents
1. Mutatiadiralyss Reagents Codons 12 and 13 User Manual V1 1 Cat No GP05 C3 TrimGen f i GENETIC DIAGNOSTICS 32 reactions trimgen com TrimGen Mutector KRAS GP05 C3 CONTENTS Introduction 4 Overview of Mutector Assay 5 Materials Provided 6 Materials Required 7 Equipment Required 7 DNA Sample Preparation 8 Sequencer Setup 8 Thermal Cycling Programs 9 Mutector Assay Protocol 10 A PCR Amplification 10 B PCR Product Clean Up 11 C EM Reaction 12 D EM Product Clean up 14 E Sample Loading 14 F Data Analysis 15 Storage Upon receipt of the kit store at 20 C until use At this temperature the reagents are stable for 6 months After first use store all of reagents at 2 8 C and keep them protected from direct light At this condition the reagents are stable for 1 month TOc TET TTA SO0OdD TrimGen Mutector KRAS GP05 C3 Notice to Purchaser The Mutector 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 TRIMGEN 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 TRIMGEN BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR2. 37 C 25 min 95 C 5 min Hold at 4 C 1 cycle 94 C 4min 20 cycles 94 C 45 sec 60 C 20 sec 70 C 20 sec Hold at 4 C TrimGen Mutector KRAS GP05 C3 Mutector Assay Protocol A PCR Amplification Thaw all reagents and keep on ice Spin down the reagents before use KRAS Controls Codon 12 C12 and KRAS Controls Codon 13 C13 are required for each test run A negative control water is recommended to run with the samples each time A 1 Prepare PCR Reaction Mix Master Mix 18 x 3 x 1 1 of Samples KRAS PCR Primers 1 x 3 x1 1 of Samples For negative and KRAS controls C12 amp C13 For pipetting error Transfer entire volume of the reagents to one tube and gently mix avoid bubble the contents This is the PCR Reaction Mix A 2 Collect 0 2 ml PCR strip tubes and label the tubes as follows Neg Negative control water C12 KRAS Controls Codon 12 C13 KRAS Controls Codon 13 A 3 Transfer 19ul of PCR Reaction Mix into all of the tubes A 4 Add 2ul of nuclease free water to the Neg tube A 5 Add 2ul of KRAS Controls Codon 12 to the C12 tube 10 TrimGen Mutector KRAS GP05 C3 A 6 Add 2ul of KRAS Controls Codon 13 to the C13 tube A 7 Add 1 2ul of sample DNA 20 80 ng ul to each sample tube When using TrimGen WaxFree kit for paraffin sample DNA extraction add 0 5 1ul final extract to each sample tube i
3. The peak size between the control and the sample panel may slightly shifted due to migration differences between individual capillary tubes Example The following data derived from Genetic Analyzer 3130 using 36 cm capillary array and POP 7 polymer Mutations in Codon 12 Wild Type Wild Type GGT gt GAT GGT gt AGT mutation mutation Wild Type GGT gt GCT mutation GGT gt CGT mutation GGT gt GTT GGT TGT Wild Type x mutation mutation 17 Wild Type Wild Type TrimGen Mutector KRAS GP05 C3 Mutations in Codon 13 GGC gt GAC mutation GGC gt GCc mutation GGC gt GTC mutation Wild Type GGC gt AGC mutation Wild Type m Wild Type va Wild Type GGC gt CGc mutation Sr rE aa a e e hemener arn s mi ii mpm am arr i n i I SASS Wild Type Wild Type ae GGC gt TGC mutation rl ora Lt ed Se a a a re re E 18 TA LA T IZ LL ETOZ
4. Tube EM13 EM13 mix for codon 13 KRAS EM 13 11 x 2 X1 1 of Samples KRAS DP 13 2 x 2 x1 1 of Samples For Controls Add the reagents to the tube and mix gently C 2 Collect 0 2 ml strip tubes 2 tubes per sample and 4 control tubes for each test run Label the tubes as follows One set for codon 12 EM12 set and another set for codon 13 EM13 set C up treated Controls EM12 set gt VOOD 3 EM13 set Nes c18 1 2 3 a C up treated PCR Samples 12 TrimGen C 3 C 4 C 5 C 6 C 7 C 8 C 9 Mutector KRAS GP05 C3 Transfer 13ul of EM12 mix from step C 1 into all tubes in EM12 set Transfer 13ul of EM13 mix from step C 1 into all tubes in EM13 set Add 2ul of C up treated Negative PCR control to the Neg tube in both EM12 and EM13 sets Add 2ul of C up treated C12 from step B 5 to the C12 tube Add 2ul of C up treated C13 from step B 5 to the C13 tube Add 2ul of C up treated Sample from step B 5 to each corresponding sample tube in both EM12 and EM13 sets Mix the contents and spin all tubes C 10 Place the tubes into the thermal cycler and perform EM reaction using Program 3 Program 3 1 cycle 94 C 4 min 94 C 45 sec 60 C 20 sec 70 C 20 sec Hold at 4 C 20 cycles During the EM reaction prepare step D1 D3 13 TrimGen Mutector KRAS GP05 C3 D EM Product Clean up Fil
5. Add too much sample may cause an inhibition of PCR reaction A 8 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 Optional The PCR product can be verify by agarose gel 5 ul loading The correct band size is 120 bp The procedure can be temporarily stopped after Program 1 The PCR products can be stored at 4 C for 2 3 days During the PCR prepare step B1 B2 PCR Products Clean Up B 1 Collect 0 2 ml strip tubes one tube for each PCR reaction Label the tubes the same as the PCR tubes B 2 Add 11ul of Clean up Enzymes C UP to each new tube B 3 Transfer 4ul of PCR products to each tube the remaining PCR products can be stored at 20 C for re test B 4 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 11 TrimGen Mutector KRAS GP05 C3 During the incubation prepare step C1 C4 C Mutation Enrichment and Detection EM reaction C 1 Collect two 2 ml tubes and label one tube with EM12 and the other tube with EM13 Prepare EM mix as follows Tube EM12 EM12 mix for codon 12 KRAS EM 12 11 x 2 X1 1 of Samples KRAS DP 12 2 x 2 x1 1 of Samples For Controls Add the reagents to the tube and mix gently
6. Mutector KRAS GP05 C3 M Assay PCR amplification 1 5 hours Time varies by thermal cycler used PCR product clean up 30 min EM reaction Mutation enrichment and detection 45 min Time varies by thermal cycler used EM products cleanup 5 min Capillary Electrophoresis Fragment analysis 30 40 min TrimGen Materials Provided Mutector KRAS GP05 C3 The Mutector KRAS Mutation Differentiation kit contains reagents for 32 tests Materials Master Mix KRAS PCR Primers Clean up Enzymes KRAS EM 12 KRAS EM 13 KRAS DP 12 KRAS DP 13 KRAS Control Codon 12 KRAS Control Codon 13 Loading Buffer TF 50 Filters Collection Tubes Quantity Description 650 Reagents for DNA amplification 50 PCR primer mix for amplification of KRAS gene Enzyme mix for cleanup of PCR 430 products Pre mixed reagents for enrichme 430 nt and detection of codon 12mut ations Pre mixed reagents for enrichme 430 nt and detection of codon 13mut ations 80 Pre mixed detection primers for codon 12mutations 80 Pre mixed detection primers for codon 13mutations 50 Mutation controls for codon 12 50 Mutation controls for codon 13 Sample loading buffer with size 1000 1x2 standards 64 Filter tip for reaction clean up 64 Collection tube for the TF 50 filter Light sensitive Keep these reagents protected from direct light TrimGen Mutector KRAS GP05 C3 TF 50 Filter Filter tip to
7. 0 80 ng ul The DNA concentration calculated using classical UV method measure OD may give incorrect DNA concentration A fluorescent method that directly measure intact DNA such as pico green method is recommended When using TrimGen s FFPE 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 tests for data analysis GeneMapper Analysis Step I GeneMapper Setup www trimgen com docs Partl GeneMapper Setup padf Step II 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 TrimGen Mutector KRAS GP05 C3 1 Important Spectral calibration is required before running the test 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 spectral channels to collect 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 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 Clean up
8. 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 TRIMGEN IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES 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 License The purchase of Mutector kit includes a limited nonexclusive license to use the kit _ This license does not grant rights to reproduce or modify the Mutector kit for resale or to use the Mutector 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 Trademarks The trademarks mentioned herein are the property of TrimGen or their respective owners TrimGen Corporation All rights reserved Information in this d
9. ocument is subject to change without notice TrimGen KRAS GP05 C3 manual 11 2013 TrimGen Mutector KRAS GP05 C3 Introduction Mutector KRAS kit is designed to detect 12 mutations occurring in codons 12 and 13 of KRAS gene Codon 12 Codon 13 Gly12Ser GGT gt AGT Gly13Ser GGC gt AGC Gly12Arg GGT gt CGT Gly13Arg GGC gt CGC Gly12Cys GGT gt TGT Gly13Cys GGC gt TGC Gly12Asp GGT gt GAT Gly13Asp GGC gt GAC Gly12Ala GGT gt GCT Gly13Ala GGC gt GCC Gly12Val GGT gt GTT Gly13Val GGC gt GTC The kit uses Shifted Termination Assay STA technology to enrich the mutation signal and is able to accurately detect low level somatic mutations Shifted Termination Assay STA Shifted Termination Assay is a proprietary technology that uses uniquely designed primers mixtures of modified enzymes and specially synthesized nucleotides STA technology extends primers by multiple bases to increase signal strength and fragment size creating mutation peaks that are easily distinguished from wild type The enriched mutation signals are then detected by fragment analysis The STA technology can detect low level mutations often missed by sequencing Wild type F Mutation Wild type Mutant a STA reaction Fragment analysis TrimGen Overview of Mutector if codon 12 codon 13 ms codon 12 codon 13 Wild type i Godon 13 GGCGCC mutation Mutation Wild type
10. odon 12 8 peaks will be presented as follows Any peaks that do not match the peaks in this panel are to be disregarded Two GTT mutation peaks are shown in this panel one peak 2 is next to and has the same color as the wild type peak When the resolution of capillary or polymer is low the mutant peak may merge with the wild type peak which makes it difficult to identify the mutation To overcome this issue a special primer is designed to generate the other peak 8 with a different size and color 15 TrimGen Mutector KRAS GP05 C3 Results for KRAS Controls Codon 13 7 peaks will be presented as follows GGC gt TGC black Wild Type black GGC gt AGC red Any peaks that not match the peaks in this panel are disregarded The pattern size or position of the peaks may vary slightly depending on instrument polymer type and the length of capillary Customer should validate the correct size for each peak by using the KRAS Controls Codon 12 and 13 16 TrimGen Mutector KRAS GP05 C3 Sample Analysis The wild type peak is a black peak on the right largest peak size The mutation s will show as additional peak s All mutation peaks are smaller than the wild type The peak size and color of the mutation peaks in the KRAS Controls Codon 12 and 13 are used as references to identify mutations in the sample Any peaks that do not match the correct size and color are not considered as mutations
11. remove free fluorescent dyes Store the filters at 2 8 C If the buffer inside the tip evaporated or completely dried add 200ul deionized water soak Buffer the gel overnight The tip is ready to use lt Snap off line Before spin the filter tip snap off bottom of the tip Good Dry Dried Materials required 0 2 ml PCR tubes 8 well strip tube DS 32 Matrix Standard Kit for calibration of sequencer Applied Biosystems Pat No 4345831 Equipment required Thermal Cycler Any type of thermal cycler with a 0 2 ml tube block is acceptable for performing the assay Sequencer Applied Biosystems Genetic Analyzer Instrument Data Collection Genetic analyzer 3100 Data Collection S oftware 7 Genetic analyzer 3700 V3 0 or v3 1 Genetic analyzer 3130 3500 Data Collection Genetic analyzer 3500 Sanwarevil i Data Analysis GeneMapper Software v4 0 or v4 1 GeneMapper S oftware v4 1 TrimGen Mutector KRAS GP05 C3 DNA Sample Preparation Reagents for DNA preparation are not provided with the kit Paraffin FFPE and fresh or frozen tissue samples A kit specially designed for FFPE samples 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 adjustment When using a column or bead DNA extraction method adjust the final concentration of extracted DNA to 2
12. ter Tip Preparation D 5 D 6 D 7 E 2 E 3 Collect the TF 50 Filter Tips and Collection Tubes one set for each EM reaction Snap off the bottom portion of the filter tip ref page 7 for snap off line and put tip back to collection tube Centrifuge the TF 50 Filter Tips at 1 000 x g 8000 rpm for most tabletop centrifuge for 2 3 minutes to remove the buffer from the filters Discard the Collection Tubes and move the TF 50 Filter Tips into a new Collection Tube Label the Collection Tubes with sample ID The TF 50 Filter Tips are ready for use After the EM reaction load entire 15ul reaction mix onto the top of gel in each of pre prepared TF 50 Filter Tips Centrifuge the TF 50 Filter Tips at 1 000 x g 3000 rpm for most tabletop centrifuge for 2 3 minutes Discard the TF 50 Filter Tips The solution in the collection tubes contains cleaned EM reaction products and is ready for sample loading Sample Loading Add 15yl of the Loading buffer to each well of a sequencer adapter plate Transfer 2 4ul of the filtered EM products into each well Load the plate to sequencer and run the pre set Data Collection Program ref page 8 14 TrimGen Mutector KRAS GP05 C3 F Data Analysis The KRAS Controls Codon 12 and Codon 13 represent the mutation patterns color and size Use these controls as standards to identify the peaks present in the test samples Results for KRAS Controls C
Download Pdf Manuals
Related Search