OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
RRTPCR.SW
School/ Divisional Unit
nephrology
Initial Issue date
Current version
13-04-2009
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
Title:
Using RT-PCR or Real-time PCR
Description:
amplify and detect target mRNA molecules
Associated risk assessment title and location: RA Bench top centrifuge, RA Wsate management
Describe



the activity or process
Single strand cDNA sythesis (reverse transcript product):
Total RNA is reverse transcribed in the PCR machine using oligo dT primer/random hexamer
primer by eg 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 eg a pair of gene specific primers by TfL/Taq
DNA polymerase at 94oC 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. Use of cycling B to acquire another signal at a higher temp [3-4’C below
melt] cuts background.
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 bromophenol 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





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, real-time PCR machine
Gel Doc
Samples, tubes, tips, pipette
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Page 1 of 3
Safe Work Procedure
Date Effective: 15.5.2010
Uncontrolled document when printed
Current Version: 15.5.2010
List potential hazards and risk controls
including specific precautions required

Chemicals may be hazardous – for chemicals check MSDS and wear PPE, use fume hood if necessary, all
users should be well trained and all spills cleaned immediately and disposed in chemical waste. All liquid
chemical waste is collected into a special waste bottle. Ethidium is dangerous.
 Be careful melting agarose in microwave; loosen bottle lid before heating, monitor closely, beware
superheating and violent bubbling; face protection needed.
 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 is hazardous to eyes – use a UV protective face shield. Cover the gel when it is transferred to stop
aerosol.
 Obey gel doc signs; no gloves on keyboards. Handle gel with gloves.
Corbett PCR machine
1. Select the correct rotor for your tubes (36 or 72 well rotor),
2. Use the appropriate locking ring for each of the rotors
3. Ensure samples are secure and balanced by placing them opposite each other, placed into the
rotor properly and the locking ring is on.
4. Close the lid and prepare the software,
List emergency shutdown instructions

PCR machine, real-time PCR machine, centrifuge have “OFF” switch that can be used in emergency.

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: P PEAKE
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licensing, training - e.g. course or
instruction:
___________________________________________________________________________________________________________
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Page 2 of 3
Safe Work Procedure
Date Effective: 15.5.2010
Uncontrolled document when printed
Current Version: 15.5.2010
SWP review date: 20-04-
2013
Responsibility for SWP review:
___________________________________________________________________________________________________________
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Page 3 of 3
Safe Work Procedure
Date Effective: 15.5.2010
Uncontrolled document when printed
Current Version: 15.5.2010