Applied Biosystems KRAS Mutation Analysis Reagents. Protocol

Size: px
Start display at page:

Download "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

High-Capacity cdna Reverse Transcription Kits For 200 and 1000 Reactions

High-Capacity cdna Reverse Transcription Kits For 200 and 1000 Reactions High-Capacity cdna Reverse Transcription Kits For 200 and 1000 Reactions Protocol Copyright 2006,. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this

More information

Single Cell-to-CT Kit. Protocol

Single Cell-to-CT Kit. Protocol Single Cell-to-CT Kit Protocol For Research 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

More information

One Shot TOP10 Competent Cells

One Shot TOP10 Competent Cells USER GUIDE One Shot TOP10 Competent Cells Catalog Numbers C4040-10, C4040-03, C4040-06, C4040-50, and C4040-52 Document Part Number 280126 Publication Number MAN0000633 Revision A.0 For Research Use Only.

More information

Path-ID Multiplex One-Step RT-PCR Kit

Path-ID Multiplex One-Step RT-PCR Kit USER GUIDE Path-ID Multiplex One-Step RT-PCR Kit TaqMan probe-based multiplex one-step real-time RT-PCR detection of RNA targets Catalog Numbers 4428206, 4428207, 4440022 Publication Part Number 4440907

More information

Protocol. Introduction to TaqMan and SYBR Green Chemistries for Real-Time PCR

Protocol. Introduction to TaqMan and SYBR Green Chemistries for Real-Time PCR Protocol Introduction to TaqMan and SYBR Green Chemistries for Real-Time PCR Copyright 2008, 2010 Applied Biosystems. All rights reserved. Ambion and Applied Biosystems products are for Research Use Only.

More information

Standard Mixture. TOF/TOF Calibration Mixture. Calibration Mixture 1 (Cal Mix 1, 1:10) Calibration Mixture 1 (Cal Mix 1, 1:100)

Standard Mixture. TOF/TOF Calibration Mixture. Calibration Mixture 1 (Cal Mix 1, 1:10) Calibration Mixture 1 (Cal Mix 1, 1:100) Mass Standards Kit for Calibration of AB SCIEX TOF/TOF Instruments Protocol 1 Product Description The Mass Standards Kit includes reagents needed to test instrument function, optimize instrument parameters,

More information

Applied Biosystems 3500/3500xL Genetic Analyzer

Applied Biosystems 3500/3500xL Genetic Analyzer QUICK REFERENCE Applied Biosystems 3500/3500xL Genetic Analyzer with 3500 Series Data Collection Software 3 Catalog Number 4405186, 4405187 Pub. No. 100026299 Rev. A Note: For safety and biohazard guidelines,

More information

Platinum Multiplex PCR Master Mix

Platinum Multiplex PCR Master Mix USER GUIDE Publication Part Number 4463722 Rev. A Revision Date April 2011 For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject

More information

TaqMan Fast Advanced Master Mix. Protocol

TaqMan Fast Advanced Master Mix. Protocol TaqMan Fast Advanced Master Mix Protocol For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. APPLIED

More information

For safety information, see the ABI PRISM 310 Genetic Analyzer User s Manual (PN 903565 or 4317588).

For safety information, see the ABI PRISM 310 Genetic Analyzer User s Manual (PN 903565 or 4317588). User Bulletin ABI PRISM 310 Genetic Analyzer 01/2007 SUBJECT: Syringe Care and Maintenance Overview Safety Information Warranty Information In this User Bulletin This user bulletin describes the care,

More information

ID kit. imegen Anchovies II. and E. japonicus) DNA detection by. User manual. Anchovies species (E. encrasicolus. sequencing.

ID kit. imegen Anchovies II. and E. japonicus) DNA detection by. User manual. Anchovies species (E. encrasicolus. sequencing. User manual imegen Anchovies II ID kit Anchovies species (E. encrasicolus and E. japonicus) DNA detection by sequencing Reference: Made in Spain The information in this guide is subject to change without

More information

Megaplex Pools For microrna Expression Analysis

Megaplex Pools For microrna Expression Analysis Megaplex Pools For microrna Expression Analysis Protocol Copyright 2008, 2010 Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document

More information

TaqMan Salmonella Enteritidis Detection Kit. Egg and Environmental Testing Protocol

TaqMan Salmonella Enteritidis Detection Kit. Egg and Environmental Testing Protocol TaqMan Salmonella Enteritidis Detection Kit Egg and Environmental Testing Protocol Qualitative real-time PCR assay for the detection of Salmonella Enteritidis from pre-enriched poultry eggs and environmental

More information

PROTOCOL. BigDye Direct Cycle Sequencing Kit

PROTOCOL. BigDye Direct Cycle Sequencing Kit PROTOCOL BigDye Direct Cycle Sequencing Kit For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice.

More information

HBV Quantitative Real Time PCR Kit

HBV Quantitative Real Time PCR Kit Revision No.: ZJ0002 Issue Date: Aug 7 th, 2008 HBV Quantitative Real Time PCR Kit Cat. No.: HD-0002-01 For Use with LightCycler 1.0/LightCycler2.0/LightCycler480 (Roche) Real Time PCR Systems (Pls ignore

More information

Troubleshooting Sequencing Data

Troubleshooting Sequencing Data Troubleshooting Sequencing Data Troubleshooting Sequencing Data No recognizable sequence (see page 7-10) Insufficient Quantitate the DNA. Increase the amount of DNA in the sequencing reactions. See page

More information

Creatine Kinase (CK) Enzymatic Assay Kit Manual Catalog #: 3460-07

Creatine Kinase (CK) Enzymatic Assay Kit Manual Catalog #: 3460-07 Creatine Kinase (CK) Enzymatic Assay Kit Manual Catalog #: 3460-07 TABLE OF CONTENTS GENERAL INFORMATION... 2 Product Description... 2 Procedure Overview... 2 Kit Contents, Storage and Shelf Life... 3

More information

Agilent High Sensitivity DNA Kit Guide

Agilent High Sensitivity DNA Kit Guide Agilent High Sensitivity DNA Kit Guide Agilent High Sensitivity DNA Agilent Technologies Notices Agilent Technologies, Inc. 2009, 2013 No part of this manual may be reproduced in any form or by any means

More information

PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200

PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System) 50 preps Product #60200 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com PCR and Sequencing Reaction Clean-Up Kit (Magnetic Bead System)

More information

DNA Analyst Training Laboratory Training Manual Protocol 2.02 Clean Technique

DNA Analyst Training Laboratory Training Manual Protocol 2.02 Clean Technique DNA Analyst Training Laboratory Training Manual Protocol 2.02 Clean Technique This laboratory protocol (or part thereof) has been provided as an example of a laboratory SOP, courtesy of the Illinois State

More information

BigDye Terminator v3.1 Cycle Sequencing Kit. Protocol. DRAFT August 27, 2002 12:32 pm, 4337035A_v3.1Title.fm

BigDye Terminator v3.1 Cycle Sequencing Kit. Protocol. DRAFT August 27, 2002 12:32 pm, 4337035A_v3.1Title.fm BigDye Terminator v3.1 Cycle Sequencing Kit Protocol August 27, 2002 12:32 pm, 4337035A_v3.1Title.fm Copyright 2002, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic

More information

FOR REFERENCE PURPOSES

FOR REFERENCE PURPOSES BIOO LIFE SCIENCE PRODUCTS FOR REFERENCE PURPOSES This manual is for Reference Purposes Only. DO NOT use this protocol to run your assays. Periodically, optimizations and revisions are made to the kit

More information

UltraClean PCR Clean-Up Kit

UltraClean PCR Clean-Up Kit UltraClean PCR Clean-Up Kit Catalog No. Quantity 12500-50 50 Preps 12500-100 100 Preps 12500-250 250 Preps Instruction Manual Please recycle Version: 02212013 1 Table of Contents Introduction... 3 Protocol

More information

Archived. Gloves should be changed frequently during the analysis.

Archived. Gloves should be changed frequently during the analysis. Introduction Gloves and laboratory coats Small tools Specific clean-up and housekeeping procedures are used to help protect evidence samples from conditions and agents that might serve to destroy, deteriorate,

More information

UltraClean Forensic DNA Isolation Kit (Single Prep Format)

UltraClean Forensic DNA Isolation Kit (Single Prep Format) UltraClean Forensic DNA Isolation Kit (Single Prep Format) Catalog No. Quantity 14000-10 10 preps 14000-S 1 prep Instruction Manual Please recycle Version: 10302012 1 Table of Contents Introduction...

More information

Aurora Forensic Sample Clean-up Protocol

Aurora Forensic Sample Clean-up Protocol Aurora Forensic Sample Clean-up Protocol 106-0008-BA-D 2015 Boreal Genomics, Inc. All rights reserved. All trademarks are property of their owners. http://www.borealgenomics.com support@borealgenomics.com

More information

Genomic DNA Extraction Kit INSTRUCTION MANUAL

Genomic DNA Extraction Kit INSTRUCTION MANUAL Genomic DNA Extraction Kit INSTRUCTION MANUAL Table of Contents Introduction 3 Kit Components 3 Storage Conditions 4 Recommended Equipment and Reagents 4 Introduction to the Protocol 4 General Overview

More information

Fast MicroSeq D2 LSU rdna Fungal Identification Kit

Fast MicroSeq D2 LSU rdna Fungal Identification Kit Fast MicroSeq D2 LSU rdna Fungal Identification Kit Protocol Copyright 2007, 2010 Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this

More information

RealStar HBV PCR Kit 1.0 11/2012

RealStar HBV PCR Kit 1.0 11/2012 RealStar HBV PCR Kit 1.0 11/2012 RealStar HBV PCR Kit 1.0 For research use only! (RUO) Product No.: 201003 96 rxns INS-201000-GB-02 Store at -25 C... -15 C November 2012 altona Diagnostics GmbH Mörkenstraße

More information

CompleteⅡ 1st strand cdna Synthesis Kit

CompleteⅡ 1st strand cdna Synthesis Kit Instruction Manual CompleteⅡ 1st strand cdna Synthesis Kit Catalog # GM30401, GM30402 Green Mountain Biosystems. LLC Web: www.greenmountainbio.com Tel: 800-942-1160 Sales: Sales@ greenmountainbio.com Support:

More information

DNA Sequencing Setup and Troubleshooting

DNA Sequencing Setup and Troubleshooting DNA Sequencing Setup and Troubleshooting Lara Cullen, PhD Scientific Applications Specialist Australia and New Zealand Reviewing Sequencing Data Review the Electropherogram Review the Raw Data (Signal

More information

Intended Use: The kit is designed to detect the 5 different mutations found in Asian population using seven different primers.

Intended Use: The kit is designed to detect the 5 different mutations found in Asian population using seven different primers. Unzipping Genes MBPCR014 Beta-Thalassemia Detection Kit P r o d u c t I n f o r m a t i o n Description: Thalassemia is a group of genetic disorders characterized by quantitative defects in globin chain

More information

ab185916 Hi-Fi cdna Synthesis Kit

ab185916 Hi-Fi cdna Synthesis Kit ab185916 Hi-Fi cdna Synthesis Kit Instructions for Use For cdna synthesis from various RNA samples This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 1

More information

The Techniques of Molecular Biology: Forensic DNA Fingerprinting

The Techniques of Molecular Biology: Forensic DNA Fingerprinting Revised Fall 2011 The Techniques of Molecular Biology: Forensic DNA Fingerprinting The techniques of molecular biology are used to manipulate the structure and function of molecules such as DNA and proteins

More information

Applied Biosystems 3730/3730xl DNA Analyzer

Applied Biosystems 3730/3730xl DNA Analyzer USER GUIDE Applied Biosystems 3730/3730xl DNA Analyzer GETTING STARTED GUIDE Publication Part Number 4478016 Rev. A Revision Date May 2012 For Research Use Only. Not intended for any animal or human therapeutic

More information

EZ-PAGE Electrophoresis System USER MANUAL

EZ-PAGE Electrophoresis System USER MANUAL EZ-PAGE Electrophoresis System USER MANUAL Table of Contents Safety Information.. 2 Product Description... 2 Product Contents..... 3 Specifications & Storage Conditions.. 3 Product Use..... 3 Getting Started

More information

Guidelines for Ethidium Bromide Waste Management & Disposal. University of Tennessee-Knoxville

Guidelines for Ethidium Bromide Waste Management & Disposal. University of Tennessee-Knoxville 1. Background Guidelines for Ethidium Bromide Waste Management & Disposal University of Tennessee-Knoxville Ethidium bromide (3,8 diamino-5-ethyl-6-phenyl phenanthridinium bromide, dromilac, CAS #1239-45-8),

More information

Fast SYBR Green Master Mix. Protocol

Fast SYBR Green Master Mix. Protocol Fast SYBR Green Master Mix Protocol Copyright 2007, 2010 Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to

More information

ZR DNA Sequencing Clean-up Kit

ZR DNA Sequencing Clean-up Kit INSTRUCTION MANUAL ZR DNA Sequencing Clean-up Kit Catalog Nos. D40 & D4051 Highlights Simple 2 Minute Bind, Wash, Elute Procedure Flexible 6-20 µl Elution Volumes Allow for Direct Loading of Samples with

More information

Applied Biosystems StepOne and StepOnePlus Real-Time PCR Systems

Applied Biosystems StepOne and StepOnePlus Real-Time PCR Systems Getting Started Guide Applied Biosystems StepOne and StepOnePlus Real-Time PCR Systems Relative Standard Curve and Comparative C T Experiments Getting Started Guide Get Started Applied Biosystems StepOne

More information

RealLine HCV PCR Qualitative - Uni-Format

RealLine HCV PCR Qualitative - Uni-Format Instructions for use PCR KIT FOR EXTRACTION OF RNA AND REAL TIME PCR DETECTION KIT FOR HEPATITIS C VIRUS RNA Research Use Only Qualitative Uni Format VBD0798 48 tests valid from: December 2013 Rev11122013

More information

Scott & White Institutional Biosafety Committee Compliance Program Biohazardous Material Spill Clean-Up Procedure Policy #IBC.002

Scott & White Institutional Biosafety Committee Compliance Program Biohazardous Material Spill Clean-Up Procedure Policy #IBC.002 I. Purpose Biohazardous material usage on the Scott & White campus is regulated by the Scott & White Institutional Biosafety Committee (IBC). Those investigators choosing to perform research with biohazardous

More information

Laboratory Biosafty In Molecular Biology and its levels

Laboratory Biosafty In Molecular Biology and its levels Laboratory Biosafty In Molecular Biology and its levels Workshop 16-17 Oct..2012 Guidelines Does not mean optional Laboratory Biosafety The Laboratory Biosafety Manual is an important WHO publication

More information

Agencourt RNAdvance Blood Kit for Free Circulating DNA and mirna/rna Isolation from 200-300μL of Plasma and Serum

Agencourt RNAdvance Blood Kit for Free Circulating DNA and mirna/rna Isolation from 200-300μL of Plasma and Serum SUPPLEMENTAL PROTOCOL WHITE PAPER Agencourt RNAdvance Blood Kit for Free Circulating DNA and mirna/rna Isolation from 200-300μL of Plasma and Serum Bee Na Lee, Ph.D., Beckman Coulter Life Sciences Process

More information

QGENEFV. Real-time PCR Kit for detection of mutation G1691A in Factor V gene using the Rotor-Gene Analyzer 3000/6000/Q (Corbett Research) Cat.

QGENEFV. Real-time PCR Kit for detection of mutation G1691A in Factor V gene using the Rotor-Gene Analyzer 3000/6000/Q (Corbett Research) Cat. Real-time PCR Kit for detection of mutation G1691A in Factor V gene using the Rotor-Gene Analyzer 3000/6000/Q (Corbett Research) Cat.# G0102 Institute of Applied Biotechnologies a.s. User manual Version

More information

Guide to using the Bio Rad CFX96 Real Time PCR Machine

Guide to using the Bio Rad CFX96 Real Time PCR Machine Guide to using the Bio Rad CFX96 Real Time PCR Machine Kyle Dobbs and Peter Hansen Table of Contents Overview..3 Setup Reaction Guidelines 4 Starting up the Software 5 Setup Protocol on Software 6 Setup

More information

PCR Product Pre-Sequencing Kit

PCR Product Pre-Sequencing Kit All goods and services are sold subject to the terms and conditions of sale of the company within USB Corporation or the group which supplies them. A copy of these terms and conditions is available on

More information

Medical or Biological Waste: Storage, Treatment, Disposal and Transportation Plan

Medical or Biological Waste: Storage, Treatment, Disposal and Transportation Plan Medical or Biological Waste: Storage, Treatment, Disposal and Transportation Plan 1. Scope This program covers all departments at Wellesley College who generate medical or biological waste to include Health

More information

User Bulletin. GeneMapper Software Version 4.0. Installation Options. In This User Bulletin. Overview

User Bulletin. GeneMapper Software Version 4.0. Installation Options. In This User Bulletin. Overview User Bulletin Software Version 4.0 February 2006 SUBJECT: Installation Options In This User Bulletin Overview This user bulletin covers:............................... 2 Installation Options for the........

More information

Transformation Protocol

Transformation Protocol To make Glycerol Stocks of Plasmids ** To be done in the hood and use RNase/DNase free tips** 1. In a 10 ml sterile tube add 3 ml autoclaved LB broth and 1.5 ul antibiotic (@ 100 ug/ul) or 3 ul antibiotic

More information

PicoMaxx High Fidelity PCR System

PicoMaxx High Fidelity PCR System PicoMaxx High Fidelity PCR System Instruction Manual Catalog #600420 (100 U), #600422 (500 U), and #600424 (1000 U) Revision C Research Use Only. Not for Use in Diagnostic Procedures. 600420-12 LIMITED

More information

AmpFlSTR Identifiler Direct PCR Amplification Kit

AmpFlSTR Identifiler Direct PCR Amplification Kit USER GUIDE AmpFlSTR Identifiler Direct PCR Amplification Kit for use with: 200 reaction kit (Part no. 4467831) 1000 reaction kit (Part no. 4408580) Publication Part Number 4415125 Rev. J For Forensic or

More information

Biosafety Level 2 (BSL-2) Safety Guidelines

Biosafety Level 2 (BSL-2) Safety Guidelines BLS-4 Biosafety Level 2 (BSL-2) Safety Guidelines BSL-3 BSL-2 BSL-1 BSL-2 builds upon BSL-1. If you work in a lab that is designated a BSL-2, the microbes used pose moderate hazards to laboratory staff

More information

AxyPrep TM Mag PCR Clean-up Protocol

AxyPrep TM Mag PCR Clean-up Protocol AxyPrep TM Mag PCR Clean-up Protocol Intro The AxyPrep Mag PCR Clean-up kit utilizes a unique paramagnetic bead technology for rapid, high-throughput purification of PCR amplicons. Using this kit, PCR

More information

TaqMan Mutation Detection Assay

TaqMan Mutation Detection Assay PROTOCOL TaqMan Mutation Detection Assay Competitive Allele-Specific TaqMan PCR Publication Part Number 4467011 Rev. B Revision Date April 2012 For Research Use Only. Not intended for any animal or human

More information

ZR-96 DNA Sequencing Clean-up Kit Catalog Nos. D4052 & D4053

ZR-96 DNA Sequencing Clean-up Kit Catalog Nos. D4052 & D4053 INSTRUCTION MANUAL ZR-96 DNA Sequencing Clean-up Kit Catalog Nos. D4052 & D4053 Highlights Simple 10 Minute Bind, Wash, Elute Procedure Flexible 15-20 µl Elution Volumes Allow for Direct Loading of Samples

More information

RNA Extraction and Quantification, Reverse Transcription, and Real-time PCR (q-pcr)

RNA Extraction and Quantification, Reverse Transcription, and Real-time PCR (q-pcr) RNA Extraction and Quantification, Reverse Transcription, and Real-time Preparation of Samples Cells: o Remove media and wash cells 2X with cold PBS. (2 ml for 6 well plate or 3 ml for 6cm plate) Keep

More information

Application Guide... 2

Application Guide... 2 Protocol for GenomePlex Whole Genome Amplification from Formalin-Fixed Parrafin-Embedded (FFPE) tissue Application Guide... 2 I. Description... 2 II. Product Components... 2 III. Materials to be Supplied

More information

User Manual. CelluLyser Lysis and cdna Synthesis Kit. Version 1.4 Oct 2012 From cells to cdna in one tube

User Manual. CelluLyser Lysis and cdna Synthesis Kit. Version 1.4 Oct 2012 From cells to cdna in one tube User Manual CelluLyser Lysis and cdna Synthesis Kit Version 1.4 Oct 2012 From cells to cdna in one tube CelluLyser Lysis and cdna Synthesis Kit Table of contents Introduction 4 Contents 5 Storage 5 Additionally

More information

HBV PCR detection Kit USER MANUAL

HBV PCR detection Kit USER MANUAL For professional use only HBV PCR detection Kit (PREP-NA DNA/RNA Extraction Kit included) USER MANUAL "DNA-Technology, Research & Production" LLC Russia, 142281, Moscow Region, Protvino, 2 Zheleznodorozhnaya

More information

Absolute Quantitation Using Standard Curve Getting Started Guide

Absolute Quantitation Using Standard Curve Getting Started Guide 5 cdna Reverse Primer Oligo d(t) or random hexamer Synthesis of 1st cdna strand 3 5 cdna Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantitation Using Standard Curve Getting Started

More information

Applied Biosystems 7500/7500 Fast Real-Time PCR Systems. SUBJECT: Memory Upgrade Requirements for 7500 Software v2.0

Applied Biosystems 7500/7500 Fast Real-Time PCR Systems. SUBJECT: Memory Upgrade Requirements for 7500 Software v2.0 User Bulletin Applied Biosystems 7500/7500 Fast Real-Time PCR Systems February 2008 SUBJECT: Memory Upgrade Requirements for 7500 Software v2.0 In This User Bulletin Summary This user bulletin covers:

More information

OCCUPATIONAL SAFETY AND ENVIRONMENTAL HEALTH GUIDELINE

OCCUPATIONAL SAFETY AND ENVIRONMENTAL HEALTH GUIDELINE OSEH Occupational Safety & Environmental Health OCCUPATIONAL SAFETY AND ENVIRONMENTAL HEALTH GUIDELINE Subject: Biohazardous (Medical) Waste Disposal Date: 08/19/09 Revision: 03 Page: 1 of 7 TABLE OF Section

More information

User Guide for the 21 CFR Part 11 Module in SDS Software v1.4

User Guide for the 21 CFR Part 11 Module in SDS Software v1.4 Applied Biosystems 7500/7500 Fast Real-Time PCR System User Guide for the 21 CFR Part 11 Module in SDS Software v1.4 Introduction Installation Logging in to the SDS Software Configuring the 21CFR11 Module

More information

Required Biosafety Level Three (BSL-3) Practices, Procedures, Facilities, and Safety Equipment For BSL-3 and BSL3/2 Laboratories

Required Biosafety Level Three (BSL-3) Practices, Procedures, Facilities, and Safety Equipment For BSL-3 and BSL3/2 Laboratories Required Biosafety Level Three (BSL-3) Practices, Procedures, Facilities, and Safety Equipment For BSL-3 and BSL3/2 Laboratories 1) LABORATORY OPERATIONS a) Responsibilities: A clear organization is set

More information

Applied Biosystems 7500/7500 Fast Real-Time PCR System

Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide Applied Biosystems 7500/7500 Fast Real-Time PCR System Standard Curve Getting Started Guide Get Started Applied Biosystems 7500/7500 Fast Real-Time PCR System Standard Curve Design

More information

Inc. Wuhan. Quantity Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4 Standard (liquid) 2

Inc. Wuhan. Quantity Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4 Standard (liquid) 2 Uscn Life Science Inc. Wuhan Website: www.uscnk.com Phone: +86 27 84259552 Fax: +86 27 84259551 E-mail: uscnk@uscnk.com ELISA Kit for Human Prostaglandin E1(PG-E1) Instruction manual Cat. No.: E0904Hu

More information

Beware that ordinary prescription glasses do not provide adequate protection. This is also true with poorly fitting safety glasses.

Beware that ordinary prescription glasses do not provide adequate protection. This is also true with poorly fitting safety glasses. Ethidium Bromide Introduction Ethidium bromide (EtBr) is widely used for visualization of nucleic acids in electrophoretic gels. EtBr forms fluorescent complexes, by intercalation of DNA, which are readily

More information

TaqMan MicroRNA Assays

TaqMan MicroRNA Assays TaqMan MicroRNA Assays Protocol December 23, 2005 11:22 am, mirna_title.fm Copyright 2005-6, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information

More information

Frequently Asked Questions

Frequently Asked Questions SPARK DNA Sample Prep Kit Ion Torrent (SPK0002-V08) Frequently Asked Questions Under what circumstances would I use SPARK DNA Sample Prep Kit for Ion Torrent? Enzymatics SPARK DNA Sample Prep Kit for Ion

More information

TaqMan Copy Number Assays. Protocol

TaqMan Copy Number Assays. Protocol TaqMan Copy Number Assays Protocol For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. Life Technologies

More information

TaqMan Small RNA Assays TaqMan MicroRNA Assays TaqMan sirna Assays Custom TaqMan Small RNA Assays. Protocol

TaqMan Small RNA Assays TaqMan MicroRNA Assays TaqMan sirna Assays Custom TaqMan Small RNA Assays. Protocol TaqMan Small RNA Assays TaqMan MicroRNA Assays TaqMan sirna Assays Custom TaqMan Small RNA Assays Protocol For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information

More information

Absolute Quantification Getting Started Guide

Absolute Quantification Getting Started Guide 5 cdna Reverse Primer Oligo d(t) or random hexamer Synthesis of 1st cdna strand 3 5 cdna Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide Introduction

More information

Biological Safety Program

Biological Safety Program Risk Management & Safety Main Office, Wyoming Hall Phone: (307) 766-3277 Fax: (307)766-6116 Regulated Materials Management Center Phone: (307)766-3696 Fax: (307)766-3699 Web: www.uwyo.edu/ehs Email: UWEHS@uwyo.edu

More information

Biohazardous Waste Management Plan

Biohazardous Waste Management Plan Central Michigan University Biohazardous Waste Management Plan This document has been prepared to provide guidance to Central Michigan University (CMU) employees in the use and disposal of biohazardous

More information

Biosafety Spill Response Guide

Biosafety Spill Response Guide Yale University Office of Environmental Health & Safety Biosafety Spill Response Guide Office of Environmental Health & Safety 135 College Street, 1 st Floor, New Haven, CT 06510 Telephone: 203-785-3550

More information

Applied Biosystems 7500/7500 Fast Real-Time PCR System

Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide Applied Biosystems 7500/7500 Fast Real-Time PCR System Genotyping Experiments Getting Started Guide Get Started Applied Biosystems 7500/7500 Fast Real-Time PCR System Genotyping

More information

ExpressArt Bacterial H-TR cdna synthesis kit. With extreme selectivity against rrnas

ExpressArt Bacterial H-TR cdna synthesis kit. With extreme selectivity against rrnas ExpressArt Bacterial H-TR cdna synthesis kit With extreme selectivity against rrnas suitable for 2 to 4 µg total RNA Catalogue No. 8004-A30 (30 rxns) Reagents Materials are provided for 30 cdna synthesis

More information

Sanger Sequencing and Quality Assurance. Zbigniew Rudzki Department of Pathology University of Melbourne

Sanger Sequencing and Quality Assurance. Zbigniew Rudzki Department of Pathology University of Melbourne Sanger Sequencing and Quality Assurance Zbigniew Rudzki Department of Pathology University of Melbourne Sanger DNA sequencing The era of DNA sequencing essentially started with the publication of the enzymatic

More information

SeqDirect PCR Cleaning Kit SeqDirect 96 PCR Cleaning Kit. High throughput cleaning of PCR reactions for fluorescent cycle sequencing

SeqDirect PCR Cleaning Kit SeqDirect 96 PCR Cleaning Kit. High throughput cleaning of PCR reactions for fluorescent cycle sequencing SeqDirect PCR Cleaning Kit SeqDirect 96 PCR Cleaning Kit High throughput cleaning of PCR reactions for fluorescent cycle sequencing Revision # 9903/4-100-9k14 SeqDirect PCR Cleaning Kit SeqDirect 96 PCR

More information

ABSTRACT. Promega Corporation, Updated September 2008. http://www.promega.com/pubhub. 1 Campbell-Staton, S.

ABSTRACT. Promega Corporation, Updated September 2008. http://www.promega.com/pubhub. 1 Campbell-Staton, S. A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA... A Modified Wizard SV Genomic DNA Purification System Protocol to Purify Genomic DNA from Shed Reptile Skin ABSTRACT

More information

Appendix J IBC Biohazard Spill Management Plan

Appendix J IBC Biohazard Spill Management Plan OVERVIEW Prevention is the most important part of any spill management plan. Be sure to read and understand standard operating procedures (SOP s) and protocols for safe manipulation of biohazards before

More information

Thermo Scientific DyNAmo cdna Synthesis Kit for qrt-pcr Technical Manual

Thermo Scientific DyNAmo cdna Synthesis Kit for qrt-pcr Technical Manual Thermo Scientific DyNAmo cdna Synthesis Kit for qrt-pcr Technical Manual F- 470S 20 cdna synthesis reactions (20 µl each) F- 470L 100 cdna synthesis reactions (20 µl each) Table of contents 1. Description...

More information

Revised 4-3-2012 EHS Biosafety. 1 Select appropriate containers/bags for autoclaving.

Revised 4-3-2012 EHS Biosafety. 1 Select appropriate containers/bags for autoclaving. Requirements for Decontamination by Autoclaving Summary: Steam sterilization (autoclaving) is one of the most effective methods for decontaminating biohazardous material. Autoclaves use saturated steam

More information

NIH Mammalian Gene Collection (MGC)

NIH Mammalian Gene Collection (MGC) USER GUIDE NIH Mammalian Gene Collection (MGC) Catalog number FL1002 Revision date 28 November 2011 Publication Part number 25-0610 MAN0000351 For Research Use Only. Not for diagnostic procedures. ii Table

More information

Maxwell 16 Blood DNA Purification System

Maxwell 16 Blood DNA Purification System Technical Manual Maxwell 16 Blood DNA Purification System Caution: Handle cartridges with care; seal edges may be sharp. 2800 Woods Hollow Rd. Madison, WI USA In Vitro Diagnostic Medical Device MDSS GmbH

More information

The AutoMate Express Forensic DNA Extraction System. Brad Dixon Forensic Science Applications Group

The AutoMate Express Forensic DNA Extraction System. Brad Dixon Forensic Science Applications Group The AutoMate Express Forensic DNA Extraction System Brad Dixon Forensic Science Applications Group Presentation Overview Chemistry and Instrument Overview PrepFiler Express & PrepFiler Express BTA Forensic

More information

PrimeSTAR HS DNA Polymerase

PrimeSTAR HS DNA Polymerase Cat. # R010A For Research Use PrimeSTAR HS DNA Polymerase Product Manual Table of Contents I. Description...3 II. III. IV. Components...3 Storage...3 Features...3 V. General Composition of PCR Reaction

More information

OpenArray Sample Tracker Software

OpenArray Sample Tracker Software QUICK REFERENCE OpenArray Sample Tracker Software For QuantStudio 12K Flex OpenArray Sample Block and the OpenArray Real-Time PCR System Publication Part Number 4460657 Rev. C Revision Date May 2012 Contents

More information

CopyCaller Software. User Guide

CopyCaller Software. User Guide CopyCaller Software User Guide CopyCaller Software User Guide Information in this document is subject to change without notice. For Research Use Only. Not for use in diagnostic procedures. APPLIED BIOSYSTEMS

More information

Biohazardous, Medical & Biological Waste Guidance Chart

Biohazardous, Medical & Biological Waste Guidance Chart CSULA Environmental Health and Safety Biohazardous, Medical & Biological Waste Guidance Chart The chart below provides information on how to handle most, if not all, of the items that frequently are collectively

More information

Policies. Prep Room Policies

Policies. Prep Room Policies Introduction INTRODUCTION The Microbiology Prep Room is located in 531A Life Sciences Building. The telephone number is 372-8609. It is open from 7:30 a.m. to 4:30 p.m. during the fall and spring semesters.

More information

360 Master Mix. , and a supplementary 360 GC Enhancer.

360 Master Mix. , and a supplementary 360 GC Enhancer. Product Bulletin AmpliTaq Gold 360 Master Mix and 360 DNA Polymerase AmpliTaq Gold 360 Master Mix AmpliTaq Gold 360 DNA Polymerase 360 Coverage for a Full Range of Targets AmpliTaq Gold 360 Master Mix

More information

empcr Amplification Method Manual - Lib-A

empcr Amplification Method Manual - Lib-A GS Junior Titanium Series May 2010 (Rev. April 2011) For life science research only. Not for use in diagnostic procedures. 1. WORKFLOW The emulsion-based clonal amplification (empcr amplification) of a

More information

PNA BRAF Mutation Detection Kit

PNA BRAF Mutation Detection Kit - PNA BRAF Mutation Detection Kit Catalog Number KA2102 50 tests/kit Version: 01 Intended for research use only www.abnova.com Introduction and Background Intended use The PNA BRAF Mutation Detection Kit

More information

Arcturus PicoPure RNA Isolation Kit. User Guide

Arcturus PicoPure RNA Isolation Kit. User Guide Arcturus PicoPure RNA Isolation Kit User Guide For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice.

More information

A Guide to Managing Your Biological Waste at the University at Albany

A Guide to Managing Your Biological Waste at the University at Albany A Guide to Managing Your Biological Waste at the University at Albany Section 1 - What you need to know: Definition: "Regulated Medical Waste (RMW) shall mean any of the following waste which is generated

More information

Infectious Waste Management Plan

Infectious Waste Management Plan Infectious Waste Management Plan Infectious Waste Management Plan USC Health & Safety Programs Unit 777-5269 POLICY: A. In keeping with the University of South Carolina's policy of providing protection

More information

Isolation of Total RNA from Cultured Cells

Isolation of Total RNA from Cultured Cells Isolation of Total RNA from Cultured Cells ABI PRISM 6700 Automated Nucleic Acid Workstation or 6100 Nucleic Acid PrepStation Protocol August 30, 2004 2:39 pm, CellTotalRNATitle.fm Copyright 2004, Applied

More information

NimbleGen DNA Methylation Microarrays and Services

NimbleGen DNA Methylation Microarrays and Services NimbleGen DNA Methylation Microarrays and Services Sample Preparation Instructions Outline This protocol describes the process for preparing samples for NimbleGen DNA Methylation microarrays using the

More information