Applied Biosystems ® KRAS Mutation Analysis Reagents
Protocol
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.
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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.
© 2010 Life Technologies Corporation. All rights reserved.
Part Number 4453794 Rev. A 03/2010
Contents
About This Guide . . . 5
Purpose . . . 5
Safety information . . . 5
Safety alert words . . . 5
SDSs . . . 6
PROTOCOL Applied Biosystems
®KRAS Mutation Analysis Reagents Protocol 7
Product information . . . 7Purpose of the product . . . 7
Contents and storage . . . 8
Materials and equipment required . . . 9
Workflow . . . 10
Before you begin: one-time procedures . . . 11
Set up data collection software . . . 11
Perform a spectral calibration . . . 11
Before each run . . . 12
Prepare DNA samples . . . 12
Prepare Filter Tips . . . 12
Prepare reagents . . . 12
Procedure . . . 13
Amplify DNA . . . 13
Clean up PCR products . . . 14
Perform primer extension reaction . . . 15
Remove free fluorescent dyes . . . 16
Capillary electrophoresis and fragment analysis . . . 17
Perform capillary electrophoresis . . . 17
Perform data analysis . . . 19
Troubleshooting . . . 23
APPENDIX A PCR Good Laboratory Practices . . . 25
Contents
APPENDIX B Safety . . . 27
Chemical safety . . . 28
General chemical safety . . . 28
SDSs . . . 29
Chemical waste safety . . . 29
Biological hazard safety . . . 31
General safety alerts for all chemicals . . . 31
Documentation and Support . . . 33
Related documentation . . . 33
Obtaining support . . . 33
Index . . . 35
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 www.appliedbiosystems.com/kras
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.
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.
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:
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.
Codon 12 Codon 13
Gly12Ser (GGT>AGT) Gly13Ser (GGC>AGC) Gly12Arg (GGT>CGT) Gly13Arg (GGC>CGC) Gly12Cys (GGT>TGT) Gly13Cys (GGC>TGC) Gly12Asp (GGT>GAT) Gly13Asp (GGC>GAC) Gly12Ala (GGT>GCT) Gly13Ala (GGC>GCC) Gly12Val (GGT>GTT) Gly13Val (GGC>GTC)
STA reaction Fragment analysis
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 4452080)
Components Storage Conditions
KRAS PCR Primers 50 µL Upon receipt, store all Applied Biosystems® KRAS
Mutation Analysis Reagents at −15 °C to −25 °C until use.
After first use, store all reagents at 2 to 8 °C and keep indicated reagents protected from direct light.
Under these conditions, the reagents are stable for 3 months.
DNA Amplification Master Mix 1000 µL
Clean-up Enzyme Mix 430 µL
KRAS Enrichment Mix, Codon 12†
† Light sensitive: Keep these reagents protected from direct light.
430 µL
KRAS Enrichment Mix, Codon 13† 430 µL
KRAS Detection Primers, Codon 12 80 µL
KRAS Detection Primers, Codon 13 80 µL
KRAS Mutation Controls, Codon 12 (pre-mixed mutant and wild-type DNA)
50 µL
KRAS Mutation Controls, Codon 13 (pre-mixed mutant and wild-type DNA)
50 µL
Loading Buffer† (loading buffer contains fluorescent-labeled size standards)
2 tubes, 1200 µL each
KRAS TF-50 Filter Tips 64 tips
Collection Tubes 128 tubes
Applied Biosystems® KRAS Mutation Analysis Reagents Protocol Materials and equipment required
Materials and equipment required
Table 1 For PCR
Table 2 For Capillary Electrophoresis with a 3500/3500xL Genetic Analyzer
Table 3 For Capillary Electrophoresis with a 3130/3130xl Genetic Analyzer
Item Applied Biosystems Part Number
Thermal cycler with a 0.2-mL tube block such as:
• Veriti® 96-Well Thermal Cycler
• 96-Well GeneAmp® PCR System 9700
Contact your Applied Biosystems sales representative
MicroAmp® 8-Tube Strip, 0.2 mL or
MicroAmp® Optical 96-Well Reaction Plate
PN N8010580
PN N8010560
Item Applied Biosystems Part Number
3500 Data Collection Software v1.0 Contact your Applied Biosystems
sales representative GeneMapper® Software v4.1
DS-32 Matrix Standard (Dye Set F) Matrix Standard Kit PN 4345831 Either 0.2-mL tubes or 96-well plates plus accessories:
For 0.2-mL tubes MicroAmp® 8-Tube Strip, 0.2 mL PN N8010580
8-Tube Retainer and Base Set (Standard) for 3500/3500xL Genetic Analyzers
PN 4410231
8-Strip Septa for 3500/3500xL Genetic Analyzers PN 4410701 For 96-well plates MicroAmp® Optical 96-Well Reaction Plate PN N8010560
96-Well Retainer & Base Set (Standard) for 3500/3500xL Genetic Analyzers
PN 4410228
Item Applied Biosystems Part Number
Data Collection Software v3.0 or v3.1 Contact your Applied Biosystems
sales representative GeneMapper® Software v4.0 or v4.1
DS-32 Matrix Standard (Dye Set F) Matrix Standard Kit PN 4345831 96-well plates plus accessories:
MicroAmp® Optical 96-Well Reaction Plate 96-Well Plate Retainer
96-Well Plate Base 96-Well Plate Septa
PN N8010560 PN 4317241 PN 4317237 PN 4315933
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)
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 www.appliedbiosystems.com/kras
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 4345831). 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 Before each run
Before each run
Prepare DNA samples
Extract DNA
DNA preparation reagents are not included with the Applied Biosystems® KRAS Mutation Analysis Reagents.Adjust DNA concentration
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.
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
40 94 °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 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 G5218-02). 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:
Note: During incubation, you can perform steps 1 to 4 of the “Perform primer extension reaction” procedure.
Number of Cycles Temperature Time
1 37 °C 45 minutes
1 90 °C 15 minutes
HOLD 4 °C N/A
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) × 1.1 4. 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 Procedure
7. Place the PCR tubes in a thermal cycler and run Program 3:
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.
Number of Cycles Temperature Time
1 94 °C 4 minutes
20 94 °C 45 seconds
60 °C 20 seconds
70 °C 20 seconds
HOLD 4 °C N/A
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
www.appliedbiosystems.com/kras 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 18.
4. (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 Capillary electrophoresis and fragment analysis
Figure 1 8-Tube Strip standard assembly; assemble in order shown (1 through 6)
6) Plate retainer
5) Septa
3) Well
2) 96-Well tray
1) Plate base
4) Retainer
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
www.appliedbiosystems.com/kras 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:
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
2 1 3 (red) 4 (blue) 5 (black)
6 7
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:
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
1 2
3 4 5
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
Applied Biosystems® KRAS Mutation Analysis Reagents Protocol Capillary electrophoresis and fragment analysis
Table 4 Example results for codon 12
Peak Number Peak Color Peak Size†
† Approximate peak size. Due to migration variation among different instruments, these peak sizes are not necessarily representative of the peaks you will obtain.
Interpretation
1 Black 50.54 Wild type (GGT)
2 Black 50.09 GGT>GTT
3 Red 48.60 GGT>GAT
4 Blue 48.51 GGT>GCT
5 Black 48.41 GGT>TGT
6 Blue 46.72 GGT>CGT
7 Red 46.13 GGT>AGT
GGT>GTT Wild Type
GGT>GCT
Wild Type Wild Type
GGT>GAT
GGT>TGT
Wild Type
Wild Type
GGT>CGT
Wild Type
GGT>AGT
Applied Biosystems® KRAS Mutation Analysis Reagents Protocol Capillary electrophoresis and fragment analysis
Table 5 Example results for codon 13
Peak Number Peak Color Peak Size† Interpretation
1 Black 60.20 Wild type
2 Blue 55.84 GGC>CGC
3 Red 52.12 GGC>AGC
Wild Type
GGC>GAC
Wild Type
GGC>AGC
Wild Type GGC>CGC
Wild Type GGC>GCC
Wild Type
GGC>TGC
Wild Type GGC>GTC
Applied Biosystems® KRAS Mutation Analysis Reagents Protocol Troubleshooting
Troubleshooting
Observation Possible cause Recommended action
The fluorescent signal is too strong Too much DNA has been injected into the capillaries.
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.
Background noise 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.
Perform the primer extension reaction and capillary electrophoresis with a new sample from the same source.
Extra peaks that do not match any of the peaks from the controls
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 non- existent 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.
Disregard any peaks that do not match the control peaks; these are not considered to be mutation signals.
Small mutation peaks that are difficult to identify as mutation signals
Trace amounts of mutated DNA. 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 Troubleshooting
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).
Appendix A PCR Good Laboratory Practices
APPENDIX B
Safety
This appendix covers:
■ Chemical safety . . . 28
General chemical safety . . . 28
SDSs . . . 29
Chemical waste safety . . . 29
Biological hazard safety. . . 31
■ General safety alerts for all chemicals . . . 31
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 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.
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
Appendix B Safety Chemical safety
SDSs
About SDSs
Chemical manufacturers supply current Safety Data Sheets (SDSs) with shipments of hazardous chemicals to new customers. They also provide SDSs with the firstshipment of a hazardous chemical to a customer after an SDS has been updated. SDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely.
Each time you receive a new SDS packaged with a hazardous chemical, be sure to replace the appropriate SDS in your files.
Obtaining SDSs
The SDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day. To obtain SDSs:
1. Go to www.appliedbiosystems.com, click Support, then select SDS.
2. In the Keyword Search field, enter the chemical name, product name, SDS part number, or other information that appears in the SDS of interest. Select the language of your choice, then click Search.
3. Find the document of interest, right-click the document title, then select any of the following:
• Open – To view the document
• Print Target – To print the document
• Save Target As – To download a PDF version of the document to a destination that you choose
Note: For the SDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer.
Chemical waste safety
Chemical waste hazards
CAUTION! HAZARDOUS WASTE. Refer to Safety Data Sheets and local regulations for handling and disposal.
WARNING! CHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury, illness, or death.
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 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.
Appendix B Safety Chemical safety
Chemical waste safety guidelines
To minimize the hazards of chemical waste:
• Read and understand the Safety Data Sheets (SDSs) provided by the
manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste.
• Provide primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.)
• 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.
• Handle chemical wastes in a fume hood.
• After emptying a waste container, seal it with the cap provided.
• Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations.
Waste disposal
If potentially hazardous waste is generated when you operate the instrument, you must:• Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations.
IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply.
Appendix B Safety General safety alerts for all chemicals
Biological hazard safety
General biohazard
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices).Individuals should be trained according to applicable regulatory and company/
institution requirements before working with potentially infectious materials.
Read and follow the applicable guidelines and/or regulatory requirements in the following:
• U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (stock no. 017-040- 00547-4; bmbl.od.nih.gov)
• Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR§1910.1030; www.access.gpo.gov/ nara/cfr/waisidx_01/
29cfr1910a_01.html).
• Your company’s/institution’s Biosafety Program protocols for working with/
handling potentially infectious materials.
Additional information about biohazard guidelines is available at:
www.cdc.gov
General safety alerts for all chemicals
Avoid contact with skin, eyes, and/or clothing. Read the SDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
Appendix B Safety
General safety alerts for all chemicals
Documentation and Support
Related documentation
Portable document format (PDF) versions of this guide and the following related document are available at www.appliedbiosystems.com/kras
Note: For additional documentation, see “Obtaining support” on page 33.
Obtaining support
For the latest services and support information for all locations, go to:
www.appliedbiosystems.com
At the Applied Biosystems web site, you can:
• Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities.
• Search through frequently asked questions (FAQs).
• Submit a question directly to Technical Support.
• Order Applied Biosystems user documents, SDSs, certificates of analysis, and other related documents.
• Download PDF documents.
• Obtain information about customer training.
• Download software updates and patches.
Document Part
number Description
Applied Biosystems® KRAS Mutation Analysis Reagents Software Setup and Data Analysis User Guide
4455374 Provides step-by-step procedures for setting up the Data Collection Software and GeneMapper® Software and for performing data analysis.
Documentation and Support Obtaining support
Index
Numerics
8-tube strip, assembling for capillary electrophoresis 18
A
agarose gel 14 amplify DNA 13
Applied Biosystems, contacting 33 Applied Biosystems® KRAS Mutation
Analysis Reagents contents 8
product information 7 storage conditions 8 supported software 9
B
background noise, troubleshooting 23 band size 14
biohazardous waste, handling 31
C
capillary electrophoresis 17 CAUTION, description 5 chemical safety 28
chemical waste safety 29
,
30 Clean-up Enzyme Mix 14contents, Applied Biosystems® KRAS Mutation Analysis Reagents
contents 8
controls, required per run 13
D
DANGER, description 5 data analysis
detailed instructions for 33 overview 19
Data Collection Software
detailed instructions for using 33 setup 11
detected mutations 7 detection primers 15
diluting primer extension products 17 DNA
amplification 13 concentration 12 extraction 12
DNA Amplification Master Mix 13 documentation
obtaining additional 33 related 33
DS-32 Matrix Standard 11 dye set 11
E
electrophoresis
loading volume, adjusting 17 performing 17
enrichment mix 15
equipment, not included 9 extra peaks, troubleshooting 23 extract DNA 12
F
filter tips, preparing 12 fluorescent dyes, clean-up 16
G
GeneMapper® Software
detailed instructions for using 33 guidelines
chemical safety 28
chemical waste disposal 29 chemical waste safety 30
Index
H
hazards. See safety
I
IMPORTANT, description 5 interpretation guidelines 20
L
labeling strip tubes for amplification 13
for PCR product clean-up 14 for primer extension 15 loading buffer 17
M
materials, not included 9 matrix standard 11 migration variation 21 MSDSs
about 6 description 29 obtaining 29
,
33 mutation controlsdetermining peak sizes 19 peak color and size 19 mutations detected 7
O
obtain support 33
P
PCR
controls 13 primers 13
product clean-up 16
product clean-up thermal cycling conditions 14
R
radioactive waste, handling 30 reagents, prepare before use 12
S
safety
biological hazards 31 chemical 28
chemical waste 29 guidelines 28
,
29,
30 SDSs, obtaining 33set up Data Collection Software 11 shifted terminal assay 7
signal too strong, troubleshooting 23 small mutation peaks, troubleshooting 23 software, supported for use with
Applied Biosystems® KRAS Mutation Analysis Reagents 9
spectral calibration, perform 11 STA 7
storage conditions
Applied Biosystems® KRAS Mutation Analysis Reagents 8
temporary after PCR 14
temporary for primer extension products 16 strip tubes
label for amplification 13
label for PCR product clean-up 14 label for primer extension 15
T
Technical Support, contacting 33 TF-50 filter tips
preparing 12 using 16
thermal cycling conditions 13
,
14,
16 training, information on 33troubleshooting
Index
V
variation in migration 21 verify PCR 14
W
WARNING, description 5 waste disposal, guidelines 30 waste profiles, description 30
