OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS GBD SWP 017
School/ Divisional Unit
School of Medical Sciences
Initial Issue date
13-03-2009
Current version
1.0
Current Version
Issue date
20-04-2010
Next review date
20-04-2013
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this
form.
Safe Work Procedure Title and basic description
Using RT-PCR or Real-time PCR
Title:
Description: amplify and detect target mRNA molecules
Associated risk assessment title and location: RA Bench top centrifuge 003, RA Wsate management 015 located in room 209B
Describe the activity or process
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Single strand cDNA sythesis (reverse transcript product):
Total RNA is reverse transcribed in the PCR machine using oligo dT primer/random hexamer primer by SuperScript II/Amv
reverse transcriptase at 37-48oC for 60min and then increase temperature to 94oC for 2min to stop reaction (promega Kit),
or using oligo dT primer/random hexamer primer by SuperScript III at 65oC for 5min denature, then 25oC for 20min, 50oC
for50min and 85oC for 5min to stop the reaction (Invitrogen Kit).
PCR or real-time PCR:
cDNA is amplified in the PCR machine using a pair of gene specific primers by TfL/Taq DNA polymerase at 94 oC for 2min
and 25-35 cycles of 94oC for 30sec, 55oC for 1min and 70oC for 1min, followed by a final extension at 70oC for 10min
(Promega Kit, PCR), or amplified in real-time PCR machine using a pair of gene specific primers by Taq DNA polymerase
plus SYBR Green solution at 95oC for 2min and 40 cycles of 94oC for 15sec, 60oC for 15sec and 68oC for 20sec, followed
by a additional melting curve step (Quantum scientific Kit, real-time PCR).
Electrophoresis to visualize the PCR products:
Prepare 50x TAE buffer (Tris-Acetate-EDTA)
Heat 1g agarose in 100ml 1X TAE buffer until dissolved, then cool to 50oC
Add ethidium bromide (at a final concentration of 10g/ml) to the dissolved agarose gel and pour the gel onto a tray with a
comb the will for wells large enough to accommodate at least 20l
Assemble the gel in the tank and add enough 1X TAE buffer to cover the gel by a few millimeters, then remove the comb
Add 1l load dye the PCR tubes and load PCR products to the gel
Electrophorese at 80-110 V until the bromophenal blue (the faster – migrating dye) has migrated at least 2-3 cm into the
gel. Visualize the gel on the UV transiluminator, filter 1 (Gel Doc).
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
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Suitable PPE
Amv reverse transcriptase and buffer (Promega), Tfl DNA polymerase and buffer (promega), Taq DNA polymerase and
buffer (promega), SuperscriptTM III First-strand Synthesis System for RT PCR (Invitrogen), RealmasterMix (quantum
Scientific), Agarose, Tris base, Acetic acid, Ethidium bromide
PCR machine, Eppendorf real-time PCR machine
Gel Doc
Samples, tubes, tips, pipette
List potential hazards and risk controls including
specific precautions required
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Chemicals may be hazardous – for chemicals check MSDS and wear PPE, use fume hood if necessary, all users are well
trained and all spills are cleaned immediately and disposed in chemical waste. All liquid chemical waste is collected into a
special waste bottle.
Sample can be contaminated – training is required for handling human tissue, wear PPE, all spills are cleaned
immediately with 70% ethanol and disposed in biological waste. Tubes, tips and gloves are disposed in biological waste.
Potential electrical hazard – do annual inspections of electrical equipment and ensure electrical safety tag is current.
UV light may be hazardous – use a UV protective face shield if it is necessary. Cover the gel when it is transferred to stop
aerosol.
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Page 1 of 2
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List emergency shutdown instructions
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PCR machine, real-time PCR machine, centrifuge have “OFF” switch that can be used in emergency.
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Shut all electrical equipment off at power point in case of emergency.
List clean up and waste disposal requirements
For chemicals:
Clean up with detergent and then with water
Dispose into chemical waste
For sample contamination:
Wipe down all spills with 70% ethanol
Dispose into biological waste
List legislation, standards and codes of practice used
in the development of the SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
Australia Dangerous Goods Code
Code of Practice for the Labeling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
Australian Standard AS2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
Australian Standard AS2243.7-1991. Safety in laboratories. Part 7: Electrical Aspects.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
Safe Work Procedure Form (OHS026)
UNSW Hazardous Waste Disposal Procedure
Supervisory approval, training, and review
Supervisor: Prof E Burcher
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licensing, training - e.g. course or instruction:
SWP review date: 20-04-2013
Responsibility for SWP review: Fei Shang
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Page 2 of 2
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007