Procedure:
School/Department:
SOP prepared by:
School of Biological Sciences
Sashi Kant and Nick Coleman (SMB), edit M Joseph
Section 1 - Personal Protective Equipment
1.
Safety glasses,
2.
Buttoned-up lab coat/lab gown
3.
Enclosed shoes
4.
Disposable nitrile gloves. Remove gloves when leaving the lab. Avoid contaminating other lab equipment with phenol traces from gloves.
5.
Keep hair tied back if long
Section 2 – Potential Hazards + Safety precautions
1.
Read the MSDS for phenol and chloroform before you being work. Know the risks of these chemicals. If you are unsure about the level of risk, or whether the risk can be controlled, work should not proceed. Know the location of spill kits, eyewashes, safety showers, fire extinguishers, fire blankets before starting work.
2.
Phenol is very hazardous in case of skin or eye contact, ingestion or inhalation.
(corrosive, irritant, toxic). The amount of tissue damage depends on length of contact. Eye contact can result in corneal damage or blindness. Skin contact can produce inflammation and blistering. Inhalation of dust will produce irritation to gastro-intestinal or respiratory tract, characterized by burning, sneezing and coughing. Skin inflammation is characterized by itching, scaling, reddening, or, occasionally, blistering.
3.
Chloroform is hazardous in case of skin contact, eye contact, or if ingested or inhaled. It is an irritant, an anaesthetic, and a suspected carcinogen.
4.
Ethanol is flammable, toxic if ingested, and irritating if it gets in the eye.
5.
(phenol/chloroform) In case of eye Contact: Check for and remove any contact lenses. Immediately flush eyes with plenty of water for at least 15 minutes. Get medical attention immediately.
6.
(phenol/chloroform) In case of skin Contact: Immediately flush skin with plenty of water for at least 15 minutes while removing contaminated clothing and shoes. Use glycerol (glycerine) as a soap. Cover the irritated skin with an emollient (eg. moisturising cream). Wash clothing / shoes before reuse. Get medical attention if phenol burns are extensive or severe.
7.
(phenol/chloroform) In case of inhalation: remove person to environment with fresh air. If not breathing, give artificial respiration. Get medical attention immediately if breathing is absent, if any breathing difficulties are apparent or if person shows any nervous symptom effects (eg. dizziness, loss of consciousness).
8.
Work in the fume hood whenever using phenol/chloroform.
9.
See also risk assessment document for phenol/chloroform.
Section 3 – Procedure
1.
Work in the fume hood at all times. The only exception to this rule would be if very small quantities of phenol/chloroform are being used (<100  l) in completely sealed tubes. If you can smell either phenol or chloroform when you are doing the work (both have a distinctive and strong smell), this is an indication that there is a problem – move to the hood, and/or check that the hood is switched on and operating properly.
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2.
For junior scientists, ensure proper instruction by coordinators/ demonstrators/ supervisors. Ensure that they understand the risks involved in his procedure, and have been provided with the proper protective equipment.
3.
Know where the nearest eyewash and safety shower are before beginning work.
4.
Make sure you are using appropriate tubes for the procedure – polypropylene is best, and the cap must be VERY tight-fitting. Do not use polycarbonate tubes – these will be dissolved by the phenol/chloroform. Glass is not recommended due to risk of breakage.
5.
Add an equal volume of phenol/chloroform/isoamyl alcohol mixture (25:24:1) to the DNA solution. Mix by shaking or vortexing until a uniform white emulsion is obtained.
6.
Centrifuge the tubes at appropriate speed and time (eg. 10,000 g, 10 min) to separate the mixture into an aqueous phase (top, contains nucleic acids), a junk layer (middle, contains denatured protein), and an organic layer
(phenol/chloroform/isoamyl; at the bottom).
7.
Transfer the top aqueous phase carefully to a new tube, being careful to avoid the middle junk layer. Discard remainder carefully into phenol/chloroform waste container. Discard tube into phenol/chloroform-contaminated solid waste container, or reuse after rinsing out phenol/chloroform residue into waste container.
8.
Add an equal volume of chloroform/isoamyl alcohol (24:1) to the aqueous phase, shake or vortex until a uniform milky emulsion forms.
9.
Centrifuge the tubes and retain aqueous phase as described above. Discard organic phase and tubes as above.
10.
Add 1/10 volume of 3 M sodium acetate to the aqueous phase, then 2 volumes of ice-cold 100% ethanol. Store at -20 C for at least 2 hours.
11.
Centrifuge to pellet nucleic acids. Discard supernatant into autoclave / culture waste (if ethanol waste is a small volume relative to overall culture waste volume), or use a dedicated ethanol waste container if large volumes involved.
Ethanol waste could be combined with phenol/chloroform waste. Do not put down sink.
12.
Add 70% ethanol to pellet, flick to resuspend pellet, allow to sit 5-10 min to dissolve excess salts. Centrifuge and discard ethanol as above. Drain on paper towel, then dry at elevated temperature and/or under vacuum to remove traces of ethanol (eg. 15 minutes at 50  C).
13.
Redissolve pellet in TE buffer or similar.
Section 4 – Disposal / Spills / Incidents
- Clean up any spills immediately.
- For small spills, use dry paper paper towel to absorb excess phenol/chloroform
(discard this along with other phenol/chloroform solid waste). Then wipe down with ethanol-soaked paper towel, then again with dry paper towel. Check that you cannot smell phenol or chloroform. If you can, repeat procedure, and clean wider area if necessary.
- For large spills, use a proper spill kit – eg. in spill station on your floor. A respirator should be worn to avoid exposure to phenol and chloroform fumes. Other workers should leave the area.
- Any incidents involving large spills (>1 L) or injuries must be reported to your supervisor and using the online incident reporting form
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- All phenol and chloroform waste must be disposed of as hazardous chemical waste
(see SOP for disposing of chemical waste)
Section 6 – Relevant Material safety data sheets
Refer to relevant MSDS on Phenol, Chloroform, Isoamyl alcohol and Ethanol
Section 7 - References
1.
Risk assessment for DNA extraction using phenol/chloroform
2.
Risk assessment and SOP for Disposal of Chemical Waste
3.
Risk assessment and SOP for Spill Cleanup
SOP Training Confirmation
By signing below, these individuals confirm that they have read and understood the SOP, and agree to always follow the instructions in this
SOP when performing this procedure.
Position Name Signature Date
Supervisor employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student employee / student
WHS Committee Approval
Representative: A. Prof Frank Seebacher Chair Safety Committee
Signature: ........................................................ Date: .....................................
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