Applied Biosystems KRAS Mutation Analysis Reagents. Protocol

Transcription

1 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

2 For Research Use 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: The Applied Biosystems KRAS Mutation Analysis Reagents 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 Applied Biosystems KRAS Mutation Analysis Reagents includes a limited, nonexclusive license to use the kit. This license does not grant rights to reproduce or modify the Applied Biosystems KRAS Mutation Analysis Reagents for resale, or to use the Applied Biosystems KRAS Mutation Analysis Reagents to manufacture commercial products without written approval of Applied Biosystems. No other license, expressed, implied or by estoppels is granted. LIMITED PRODUCT WARRANTY It is imperative that users strictly adhere to the protocol. Failure to do so will void the Applied Biosystems warranty of this product. Applied Biosystems makes no other warranties of any kind, expressed or implied, including without limitation, warranties of merchantability of fitness for a particular purpose. TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Life Technologies Corporation. All rights reserved. Part Number Rev. A 03/2010

3 Contents About This Guide Purpose Safety information Safety alert words SDSs PROTOCOL Applied Biosystems KRAS Mutation Analysis Reagents Protocol 7 Product information Purpose of the product Contents and storage Materials and equipment required Workflow Before you begin: one-time procedures Set up data collection software Perform a spectral calibration Before each run Prepare DNA samples Prepare Filter Tips Prepare reagents Procedure Amplify DNA Clean up PCR products Perform primer extension reaction Remove free fluorescent dyes Capillary electrophoresis and fragment analysis Perform capillary electrophoresis Perform data analysis Troubleshooting APPENDIX A PCR Good Laboratory Practices Applied Biosystems KRAS Mutation Analysis Reagents Protocol 3

4 Contents APPENDIX B Safety Chemical safety General chemical safety SDSs Chemical waste safety Biological hazard safety General safety alerts for all chemicals Documentation and Support Related documentation Obtaining support Index Applied Biosystems KRAS Mutation Analysis Reagents Protocol

5 About This Guide Purpose The Applied Biosystems KRAS Mutation Analysis Reagents Protocol provides detailed instructions for using the Applied Biosystems KRAS Mutation Analysis Reagents and general instructions for the complete mutation analysis workflow from sample preparation through data analysis. For detailed instructions on software setup and data analysis, refer to the Applied Biosystems KRAS Mutation Analysis Reagents Software Setup and Data Analysis User Guide at Safety information Note: For general safety information, see this section and Appendix B, Safety on page 27. When a hazard symbol and hazard type appear by a chemical name or instrument hazard, see the Safety Appendix for the complete alert on the chemical or instrument. Safety alert words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards. Each alert word IMPORTANT, CAUTION, WARNING, DANGER implies a particular level of observation or action, as defined below: IMPORTANT! Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. CAUTION! Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. WARNING! Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. DANGER! Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. Applied Biosystems KRAS Mutation Analysis Reagents Protocol 5

6 About This Guide Safety information SDSs The Safety Data Sheets (SDSs) for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day. For instructions on obtaining SDSs, see SDSs on page 29. IMPORTANT! For the SDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer. 6 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

7 PROTOCOL Applied Biosystems KRAS Mutation Analysis Reagents Protocol Product information Purpose of the product The Applied Biosystems KRAS Mutation Analysis Reagents are designed to detect and differentiate 12 mutations in codons 12 and 13 of the KRAS gene: Codon 12 Codon 13 Gly12Ser (GGT>AGT) Gly12Arg (GGT>CGT) Gly12Cys (GGT>TGT) Gly12Asp (GGT>GAT) Gly12Ala (GGT>GCT) Gly12Val (GGT>GTT) Gly13Ser (GGC>AGC) Gly13Arg (GGC>CGC) Gly13Cys (GGC>TGC) Gly13Asp (GGC>GAC) Gly13Ala (GGC>GCC) Gly13Val (GGC>GTC) Applied Biosystems KRAS Mutation Analysis Reagents allow detection of low-level somatic mutations through the Shifted Termination Assay (STA) primer extension reaction and capillary electrophoresis fragment analysis. During the primer extension reaction, uniquely designed primers, modified enzymes, and specially synthesized nucleotides produce primer extension products for wild-type and mutant-targeted sequences. The amplified extension products result in a distinct peak pattern to differentiate between wild-type and mutant alleles, which are detected through DNA sizing fragment analysis using capillary electrophoresis. STA reaction Fragment analysis Applied Biosystems KRAS Mutation Analysis Reagents Protocol 7

8 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Product information Contents and storage The Applied Biosystems KRAS Mutation Analysis Reagents provide reagents for 32 reactions, including controls: Applied Biosystems KRAS Mutation Analysis Reagents (PN ) Components Storage Conditions KRAS PCR Primers 50 µl Upon receipt, store all Applied Biosystems KRAS DNA Amplification Master Mix 1000 µl Mutation Analysis Reagents at 15 C to 25 C until use. Clean-up Enzyme Mix 430 µl After first use, store all reagents at 2 to 8 C and KRAS Enrichment Mix, Codon 12 KRAS Enrichment Mix, Codon µl 430 µl keep indicated reagents protected from direct light. Under these conditions, the reagents are stable for 3 months. KRAS Detection Primers, Codon µl KRAS Detection Primers, Codon µl KRAS Mutation Controls, Codon 12 (pre-mixed mutant and wild-type DNA) KRAS Mutation Controls, Codon 13 (pre-mixed mutant and wild-type DNA) Loading Buffer (loading buffer contains fluorescent-labeled size standards) KRAS TF-50 Filter Tips Collection Tubes Light sensitive: Keep these reagents protected from direct light. 50 µl 50 µl 2 tubes, 1200 µl each 64 tips 128 tubes 8 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

9 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Materials and equipment required Materials and equipment required Table 1 For PCR Item Thermal cycler with a 0.2-mL tube block such as: Veriti 96-Well Thermal Cycler 96-Well GeneAmp PCR System 9700 MicroAmp 8-Tube Strip, 0.2 ml or MicroAmp Optical 96-Well Reaction Plate Applied Biosystems Part Number Contact your Applied Biosystems sales representative PN N PN N Table 2 For Capillary Electrophoresis with a 3500/3500xL Genetic Analyzer Item Applied Biosystems Part Number 3500 Data Collection Software v1.0 Contact your Applied Biosystems GeneMapper Software v4.1 sales representative DS-32 Matrix Standard (Dye Set F) Matrix Standard Kit PN Either 0.2-mL tubes or 96-well plates plus accessories: For 0.2-mL tubes MicroAmp 8-Tube Strip, 0.2 ml PN N Tube Retainer and Base Set (Standard) for 3500/3500xL Genetic Analyzers PN Strip Septa for 3500/3500xL Genetic Analyzers PN For 96-well plates MicroAmp Optical 96-Well Reaction Plate PN N Well Retainer & Base Set (Standard) for 3500/3500xL Genetic Analyzers PN Table 3 For Capillary Electrophoresis with a 3130/3130xl Genetic Analyzer Item Applied Biosystems Part Number Data Collection Software v3.0 or v3.1 GeneMapper Software v4.0 or v4.1 DS-32 Matrix Standard (Dye Set F) Matrix Standard Kit PN well plates plus accessories: MicroAmp Optical 96-Well Reaction Plate 96-Well Plate Retainer 96-Well Plate Base 96-Well Plate Septa Contact your Applied Biosystems sales representative PN N PN PN PN Applied Biosystems KRAS Mutation Analysis Reagents Protocol 9

10 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Workflow Workflow Primer Extension Reaction (2.5 to 4 hours) Amplify DNA (1 to 2 hours, depending on the thermal cycler) (page 13) Clean up PCR products (1 hour) (page 14) Perform primer extension reaction (0.5 to 1 hour, depending on the thermal cycler) (page 15) Remove free fluorescent dyes (5 minutes) (page 16) Capillary Electrophoresis and Fragment Analysis (40 to 60 minutes) Perform capillary electrophoresis (page 17) Perform data analysis (page 19) 10 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

11 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Before you begin: one-time procedures Before you begin: one-time procedures Set up data collection software Before using the Applied Biosystems KRAS Mutation Analysis Reagents for the first time, set up the Data Collection Software v3.0 or v3.1, or the 3500 Data Collection software v1.0. For instructions, refer to the Applied Biosystems KRAS Mutation Analysis Reagents Software Setup and Data Analysis User Guide at Perform a spectral calibration IMPORTANT! Before using the Applied Biosystems KRAS Mutation Analysis Reagents for the first time on a genetic analyzer, run a spectral calibration to set up the correct spectral channels to read the test results. Use the DS-32 Matrix Standard (Dye Set F) Matrix Standard Kit (Applied Biosystems PN ). Refer to the DS-32 Matrix Standard Product Insert to prepare the matrix standards. Refer to the appropriate instrument user guide for instructions on performing the spectral calibration. Applied Biosystems KRAS Mutation Analysis Reagents Protocol 11

12 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Before each run Before each run Prepare DNA samples Extract DNA Adjust DNA concentration DNA preparation reagents are not included with the Applied Biosystems KRAS Mutation Analysis Reagents. When using a column or bead DNA extraction method, adjust the final concentration of extracted DNA to 20 to 80 ng/μl. Prepare Filter Tips Each run requires one KRAS TF-50 Filter Tip per reaction. Before beginning a run, examine the filter tips. If: The buffer on top of the gel has evaporated, add 100 to 150 μl deionized water to each filter to rehydrate the gel The gel is completely dry (white in appearance), soak the filter overnight in deionized water Prepare reagents Thaw all reagents and keep on ice. Spin down the reagents before use. 12 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

13 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Procedure Procedure Amplify DNA 1. To prepare the PCR reaction mix, pipette the following volumes to one tube, then vortex the contents. DNA Amplification Master Mix Volume (µl) = 26 (Number of Samples + 3) 1.1 KRAS PCR Primers Volume (µl) = 1 (Number of Samples + 3) 1.1 Note: The mix includes volume for three controls. For each PCR run: Two positive mutation controls (KRAS Codon 12 and Codon 13) are required One negative control (water) is recommended Note: The volumes are multiplied by 1.1 to provide 10% excess volume to compensate for pipetting losses. 2. Label 0.2-mL PCR strip tubes or 96-well plate wells as follows: 3. Add PCR reaction mix, controls, and samples as follows: a. Add 27 μl of PCR reaction mix to each sample and control tube. b. Add 3 μl of nuclease-free water to the Neg tube. c. Add 3 μl of KRAS Mutation Controls, Codon 12 to the M12 tube. d. Add 3 μl of KRAS Mutation Controls, Codon 13 to the M13 tube. e. Add 3 μl of extracted DNA (20 to 80 ng/μl) to each sample tube. 4. Place the PCR tubes in a thermal cycler and run Program 1: Number of Cycles Temperature Time 1 94 C 5 minutes C 30 seconds 52 C 45 seconds 72 C 45 seconds 1 72 C 5 minutes HOLD 4 C N/A Applied Biosystems KRAS Mutation Analysis Reagents Protocol 13

14 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Procedure 5. (Optional) To verify that PCR was successful, load 5 μl of the PCR products onto 1 to 2% agarose gel such as Invitrogen E-Gel 2% with SYBR Safe DNA gel stain (PN G ). The correct band size is 120 bp. Note: During PCR, you can perform steps 1 and 2 of the Clean up PCR products procedure. Note: (Optional) After PCR, you can temporarily stop the procedure. The PCR products can be stored at 4 C for 2 to 3 days or 20 C for one week. Clean up PCR products 1. Label 0.2-mL PCR strip tubes or 96-well plate wells (one for each PCR reaction, including controls) as follows: 2. Add 11 μl of Clean-up Enzyme Mix to each new tube. 3. Transfer 4 μl of PCR products to each tube (store the remaining PCR products at 20 C for re-testing). 4. Vortex the contents and spin all tubes. 5. Place the PCR tubes in a thermal cycler and incubate using Program 2: Number of Cycles Temperature Time 1 37 C 45 minutes 1 90 C 15 minutes HOLD 4 C N/A Note: During incubation, you can perform steps 1 to 4 of the Perform primer extension reaction procedure. 14 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

15 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Procedure Perform primer extension reaction 1. Label two new 2-mL tubes K12 (for the KRAS codon 12 primer extension mix) and K13 (for the KRAS codon 13 primer extension mix). 2. To prepare the KRAS codon 12 primer extension mix, pipette the following volumes to the K12 tube, then vortex the contents. KRAS Enrichment Mix, Codon 12 (µl) = 11 (Number of Samples + 2) 1.1 KRAS Detection Primers, Codon 12 (µl) = 2 (Number of Samples + 2) 1.1 Note: The mix includes volume for the positive and negative controls. Note: The volumes are multiplied by 1.1 to provide 10% excess volume to compensate for pipetting losses. 3. To prepare the KRAS codon 13 primer extension mix, pipette the following volumes to the K13 tube, then vortex the contents. KRAS Enrichment Mix, Codon 13 (µl) = 11 (Number of Samples + 2) 1.1 KRAS Detection Primers, Codon 13 (µl) = 2 (Number of Samples + 2) Label 0.2-mL PCR strip tubes or 96-well plate wells (two tubes for each PCR reaction, and four control tubes per run) as follows: 5. Add primer extension mixes and cleaned-up samples and controls as follows: a. Add 13 μl of K12 primer extension mix to each K12 sample and control tube. b. Add 13 μl of K13 primer extension mix to each K13 sample and control tube. c. Add 2 μl of cleaned-up negative PCR control to both Neg tubes. d. Add 2 μl of cleaned-up M12 positive control to the M12 tube. e. Add 2 μl of cleaned-up M13 positive control to the M13 tube. f. Add 2 μl of cleaned-up PCR products to each corresponding K12 and K13 sample tube. 6. Vortex the contents, then spin all tubes. Applied Biosystems KRAS Mutation Analysis Reagents Protocol 15

16 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Procedure 7. Place the PCR tubes in a thermal cycler and run Program 3: Number of Cycles Temperature Time 1 94 C 4 minutes C 45 seconds 60 C 20 seconds 70 C 20 seconds HOLD 4 C N/A Note: During the primer extension reaction, you can perform step 1 of the Remove free fluorescent dyes procedure. Note: (Optional) You can temporarily stop the procedure after the primer extension reaction. The extension products can be stored at 20 C for up to one week. Remove free fluorescent dyes 1. Prepare one set of KRAS TF-50 Filter Tips and Collection Tubes for each reaction (including control reactions) as follows: a. Snap off the bottom portion of each filter tip and place the filter tip into a collection tube. b. Centrifuge the assembled filter tips and collection tubes at 1000 x g (3000 rpm for most tabletop centrifuges) for 2 minutes to remove the excess liquid from the filters. c. Discard the collection tubes and place each filter tip into a new collection tube. d. Label the new collection tubes with sample IDs. The filter tips and collection tubes are ready for use. 2. Pipette 15 μl of each primer extension products onto the gel of a pre-prepared filter tip. 3. Centrifuge the assembled filter tips and collection tubes at 1000 x g (3000 rpm for most tabletop centrifuges) for 2 minutes. 4. Discard the filters. The solution in the tubes contains the primer extension products and is ready for capillary electrophoresis. 16 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

17 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Capillary electrophoresis and fragment analysis Capillary electrophoresis and fragment analysis Perform capillary electrophoresis IMPORTANT! Refer to the Applied Biosystems KRAS Mutation Analysis Reagents Software Setup and Data Analysis User Guide at to set up the Data Collection Software. 1. Add 15 μl of Loading Buffer to each tube (if using 8-tube strips) or plate well (if using a 96-well plate). 2. Add 2 to 4 μl of filtered primer extension products to each tube or plate well. Note: The resulting signal may vary depending on the genetic analyzer used. It is recommended that you adjust the loading volume of primer extension products to optimize the reaction for your genetic analyzer. If the signal is too strong, then reduce the loading volume by 0.5 to 2.0 μl, or dilute the primer extension products 2 to 5 times with water (not buffer). 3. If you are using 8-tube strips, assemble the tubes in the plate base using a plate retainer as shown Figure 1, 8-Tube Strip standard assembly; assemble in order shown (1 through 6) on page (Optional) Centrifuge the plate to bring liquid to the bottom of the tubes or plate wells and remove air bubbles. 5. Place the plate in the genetic analyzer, set up the data collection software as described in the Applied Biosystems KRAS Mutation Analysis Reagents Software Setup and Data Analysis User Guide, then link the plate and start the run. Applied Biosystems KRAS Mutation Analysis Reagents Protocol 17

18 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Capillary electrophoresis and fragment analysis Figure 1 8-Tube Strip standard assembly; assemble in order shown (1 through 6) 6) Plate retainer 5) Septa 4) Retainer 3) Well 2) 96-Well tray 1) Plate base 18 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

19 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Capillary electrophoresis and fragment analysis Perform data analysis IMPORTANT! Refer to the Applied Biosystems KRAS Mutation Analysis Reagents Software Setup and Data Analysis User Guide at to set up and perform data analysis in GeneMapper Software v4.0 or v4.1. Results for mutation controls The KRAS Mutation Controls create peaks of distinct color and size for each of the six codon 12 and six codon 13 mutations. Use these controls as a standard to identify mutation(s) present in the samples. To determine the control peak sizes: 1. The KRAS Mutation Controls, Codon 12 is genomic DNA with six mutations in codon 12. Read the seven peaks from right to left, and record the peak size in the table below: 3 (red) 4 (blue) 5 (black) Peak Number Peak Color Peak Size Interpretation 1 Black Wild type 2 Black GGT>GTT 3 Red GGT>GAT 4 Blue GGT>GCT 5 Black GGT>TGT 6 Blue GGT>CGT 7 Red GGT>AGT Applied Biosystems KRAS Mutation Analysis Reagents Protocol 19

20 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Capillary electrophoresis and fragment analysis 2. KRAS Mutation Controls, Codon 13 is genomic DNA with six mutations in codon 13. Read the seven peaks from right to left, and record the peak size in the table below: 6 (red) 7 (black) Peak Number Peak Color Peak Size Interpretation 1 Black Wild type 2 Blue GGC>CGC 3 Red GGC>AGC 4 Black GGC>TGC 5 Blue GGC>GCC 6 Red GGC>GAC 7 Black GGC>GTC Example results for samples When examining results, note that: The wild-type peak is the black peak on the right with the largest peak size. The mutations are shown as an additional peak(s). All mutation peaks are smaller than the wild type. Compare the peak size and color with the appropriate control panel. Disregard any peaks that do not match the color and size of the control panel peaks. Peak size and location may vary depending on the genetic analyzer, polymer type, and length of the capillary. Use the peaks generated by the KRAS Mutation Controls to confirm the correct peak size. Peak location may vary between individual capillaries. 20 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

21 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Capillary electrophoresis and fragment analysis Table 4 Example results for codon 12 Wild Type Wild Type GGT>GTT Wild Type GGT>GCT GGT>GAT Wild Type Wild Type Wild Type GGT>CGT GGT>AGT GGT>TGT Peak Number Peak Color Peak Size Interpretation 1 Black Wild type (GGT) 2 Black GGT>GTT 3 Red GGT>GAT 4 Blue GGT>GCT 5 Black GGT>TGT 6 Blue GGT>CGT 7 Red GGT>AGT Approximate peak size. Due to migration variation among different instruments, these peak sizes are not necessarily representative of the peaks you will obtain. Applied Biosystems KRAS Mutation Analysis Reagents Protocol 21

22 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Capillary electrophoresis and fragment analysis Table 5 Example results for codon 13 Wild Type Wild Type GGC>GAC GGC>AGC GGC>CGC Wild Type GGC>GCC Wild Type Wild Type GGC>GTC Wild Type GGC>TGC Peak Number Peak Color Peak Size Interpretation 1 Black Wild type 2 Blue GGC>CGC 3 Red GGC>AGC 4 Black GGC>TGC 5 Blue GGC>GCC 6 Red GGC>GAC 7 Black GGC>GTC Approximate peak size. Due to migration variation among different instruments, these peak sizes are not necessarily representative of the peaks you will obtain. 22 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

23 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Troubleshooting Troubleshooting Observation Possible cause Recommended action The fluorescent signal is too strong Background noise Extra peaks that do not match any of the peaks from the controls Small mutation peaks that are difficult to identify as mutation signals Too much DNA has been injected into the capillaries. For Applied Biosystems KRAS Mutation Analysis Reagents, the background is normally low. High background noise may be caused by poor quality DNA. If the PCR reaction does not perform properly, the intensity of the wild-type peak is usually lower than 300 and many small peaks are observed. Low initial DNA concentration or problems during amplification may cause PCR primers to form primer dimers. Primer dimers can produce false peaks that do not match peaks from the codon 12 and codon 13 controls. The non-matching peaks may be accompanied by a small or nonexistent wild-type peak. Some samples may contain a silent mutation at the third base that can produce peaks that do not match the M12 and M13 controls. Trace amounts of mutated DNA. To optimize the peak signal, either: Dilute the filtered primer extension products by 3 to 5 times with water or Reduce the loading volume of the filtered primer extension products to 0.5 to 1.0 μl. Perform the primer extension reaction and capillary electrophoresis with a new sample from the same source. Disregard any peaks that do not match the control peaks; these are not considered to be mutation signals. Determine the correct color and size of the suspect peak by using the tables on page 21 and on page 22, then perform the following calculation: Ratio = (area of mutant peak) / (area of wild-type peak) A ratio greater than 0.03 may indicate a mutation peak. Applied Biosystems KRAS Mutation Analysis Reagents Protocol 23

24 Applied Biosystems KRAS Mutation Analysis Reagents Protocol Troubleshooting 24 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

25 APPENDIX A PCR Good Laboratory Practices When preparing samples for PCR amplification: Use a positive-displacement pipette or aerosol-resistant pipette tips. Follow proper pipette-dispensing techniques to prevent aerosols. Wear clean gloves and a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation). Change gloves whenever you suspect that they are contaminated. Maintain separate areas and dedicated equipment and supplies for: Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area. Open and close all sample tubes carefully. Centrifuge tubes before opening. Try not to splash or spray PCR samples. Keep reactions and components capped as much as possible. Clean lab benches and equipment periodically with 10% bleach solution. Use DNAZap Solution (PN AM9890). Applied Biosystems KRAS Mutation Analysis Reagents Protocol 25

26 Appendix A PCR Good Laboratory Practices 26 Applied Biosystems KRAS Mutation Analysis Reagents Protocol

27 APPENDIX B Safety This appendix covers: Chemical safety General chemical safety SDSs Chemical waste safety Biological hazard safety General safety alerts for all chemicals Applied Biosystems KRAS Mutation Analysis Reagents Protocol 27

28 Appendix B Safety Chemical safety Chemical safety General chemical safety Chemical hazard warning WARNING! CHEMICAL HAZARD. Before handling any chemicals, refer to the Safety Data Sheet (SDS) provided by the manufacturer, and observe all relevant precautions. WARNING! CHEMICAL HAZARD. All chemicals in the instrument, including liquid in the lines, are potentially hazardous. Always determine what chemicals have been used in the instrument before changing reagents or instrument components. Wear appropriate eyewear, protective clothing, and gloves when working on the instrument. WARNING! CHEMICAL HAZARD. Four-liter reagent and waste bottles can crack and leak. Each 4-liter bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in a glass container because of the risk of breaking or shattering. Reagent and waste bottles can crack and leak. Each waste bottle should be secured in a lowdensity polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. Chemical safety guidelines To minimize the hazards of chemicals: Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See About SDSs on page 29.) Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the SDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the SDS. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer s cleanup procedures as recommended in the SDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. 28 Applied Biosystems KRAS Mutation Analysis Reagents Protocol