OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS.CGM SWP015
Initial Issue date
30/06/09
School/ Divisional Unit
SoMS / Oncology Research Unit/MDU
Current version
Current Version
Next review date
1.0
30/06/09
Issue date
30/06/09
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: Extraction of DNA Fragments from Agarose Gel
Description: To safely and correctly extract DNA fragments from agarose gels using Promega Wizard SV Gel Clean-Up system
Associated risk assessment title and location:15_RA_Gel extraction_CM
Describe the activity or process
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Take ethidium-bromide-stained agarose gel with electrophoresed DNA, and over a UV transilluminator carefully cut out
DNA fragment of interest using a scalpel.
Place agarose fragment in an eppendorf tube and add an equal volume of binding buffer (1μL per mg agarose)
Heat in 50-65°C heat block until agarose is entirely melted (approx. 10 minutes)
Transfer melted agarose solution to SV minicolumn, placed inside collection tube. Incubate for 1 min.
Spin in microcentrifuge for 1 min at top speed
Discard spin-through, and wash by adding 800μL wash buffer
Spin in microcentrifuge for 1 min at top speed
Discard spin-through, and wash by adding 500μL wash buffer
Spin in microcentrifuge for 1 min at top speed
Discard spin-through, and Spin in microcentrifuge for 1 min at top speed
Place SV minicolumn into clean eppendorf tube and add 50 μL H2O or TE buffer. Incubate for 1 min.
Spin in microcentrifuge for 1 min at top speed to collect DNA
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
Agarose gel with electrophoresed DNA
UV transilluminator
Scalpel
Eppendorf tubes
Personel protective clothing: gloves, labcoat, face shield
Promega Wizard Gel Extraction kit
Heat block
Microcentrifuge
List potential hazards and risk controls including
specific precautions required
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Exposure to UV light from transilluminator
Minimise exposure to UV light by turning the UV lamp on for a minimal amount of time, don PPE including labcoat and
gloves, and protect the face and eyes with a plastic face-shield. [See 62_SWP_UVEquipment_JH.doc]
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Exposure to Ethidium Bromide
Ethidium bromide may be harmful
Wear PPE. Minimise direct contact with gel.
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Cutting hazard when using scalpel
Be careful when using scalpel not to cut self. Always cut away from self. Wear closed-shoes in case of dropped scalpel.
Take care when exchanging scalpel blades.
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Electrical shock hazard from electrical equipment (heat block, centrifuge)
Ensure electrical devices are correctly plugged in.
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Microcentrifuge
Ensure microcentrifuge is correctly balanced. Always tighten lid before turning on.
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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
Emergency electrical shutdown button is located in laboratory near entrance
List clean up and waste disposal requirements
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Dispose of agarose gel in biohazard bin
Dispose of all buffers and supernatents in biological liquid waste
Dispose of scalpel blades in sharps bin
Dispose of Ethidium bromide waste in toxic waste bin.
Clean ethidium bromide contaminated equipment with ethanol (dissolves EtBr), or by exposure to UV light
List legislation, standards and codes of practice
used in the development of the SWP
NSW OHS Act 2000, NSW OHS Regulation 2001
Code of Practice for the Labelling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3:2002 Safety in laboratories, Part 3:Microbiological aspects and containment facilities)
AS/NZS 2243.3:2002/Amdt 1:2003 Safety in laboratories, Part 3:Microbiological aspects and containment
facilities)
AS/NZS 2243.7-1991. Safety in laboratories. Part 7: Electrical Aspects.
AS/NZS 2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
AS/NZS 1336:1997 Recommended Practices for Occupational Eye Protection
AS/NZS 2211.9:2002 Safety of laser products - Compilation of maximum permissible exposure to
incoherent optical radiation
AS/NZS 1338.2:1992 Filters for eye protectors - Filters for protection against ultraviolet radiation
Safe Work Procedure Form (OHS026
Supervisory approval, training, and review
Supervisor: Peter Gunning
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Training as per Training Needs Analysis, Induction to Lab, Training in this SWP
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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