NIU Biological Sciences Standard Operating Procedure
#1
CONTACT INFORMATION:
Procedure Title
Working with Phenol:Chloroform in Molecular Biology
Procedure Author
Barrie Bode
Date of
Creation/Revision
September 23, 2012
Name of Responsible
Person
Barrie Bode
Location of Procedure
Montgomery 412
Approval Signature
ORC#S11-0001 OR #S11-0002
#2
THIS STANDARD OPERATING PROCEDURE (SOP) IS FOR A:
Specific laboratory procedure or experiment
Generic laboratory procedure that covers several chemicals
 Generic use of specific chemical or class of chemicals with similar hazards
#3
PROCESS OR EXPERIMENT DESCRIPTION
Phenol and choloroform are widely-used chemicals for nucleic acid purification in
molecular biology. Both are listed as hazardous by OSHA and must be handled very
carefully. Phenol causes severe chemical burns upon exposure to skin; therefore,
gloves, a lab coat and safety glasses must be worn when using this chemical.
Frequency:
□ one time □ daily  weekly or  monthly
□ other:_____________
Duration per
Expt:
#4
1-2 hours
SAFETY LITERATURE REVIEW & HAZARD SUMMARY
For assistance with this form contact NIU Environmental Health and Safety, 815-753-0404.
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NIU Biological Sciences Standard Operating Procedure
Phenol causes severe chemical burns so appropriate PPE such as gloves, lab coat
and safety glasses must be worn when working with it. Likewise, chloroform is listed
as an irritant and carcinogen.
#5
STORAGE REQUIREMENTS
Phenol is stored at 4°C, usually as a saturated aqueous solution (Tris-buffered; pH
4.3 or 8.0), in a mixture with chloroform at a ratio of 25:24:1
(Phenol:chloroform:isoamyl alcohol), in a tightly-closed screw-cap amber bottle. The
bottles are placed in a tray to avoid tipping or spilling. Chloroform is also stored at
room temperature in a screw-cap bottle in the corrosives cabinet.
#6
STEP-BY-STEP OPERATING PROCEDURE
1. Don personal protective equipment.
 appropriate street clothing (long pants, close-toed shoes)
 gloves; Nitrile
□ safety goggles  safety glasses □ face shield
 lab coat
2. Check the location and accessibility of the safety equipment that serves your
lab:
ITEM
Fume Hood
Eyewash station
STATUS
Location: MO 412
Location: MO 412 on south sink
1. The bottle of phenol should be opened under the fume hood to avoid breathing
vapors.
2. Remove a pre-determined volume of the phenol:chloroform solution to a 50 ml
screw-cap tube using a pipet, or by carefully pouring it. Replace the cap and
place tube on ice. Use this as your working stock.
3. Typically, only 0.5 – 1.0 ml of phenol chloroform is used at a time in the
purification of nucleic acids. Withdraw such volumes from the 50 ml tube using a
micro pipette and disposable tips. After phenol solution has been dispensed,
discard the pipette tip into a bag designate for phenol-chloroform solid waste.
4. Purification procedures take place in 0.6 or 1.5 ml snap-cap centrifuge tubes.
Make sure that the cap is tightly secured closed prior to using the vortex mixer to
For assistance with this form contact NIU Environmental Health and Safety, 815-753-0404.
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NIU Biological Sciences Standard Operating Procedure
blend the solutions. Check your gloves often to ensure that no residual phenol
contaminates them.
5. All centrifugation steps are carried out in a closed micro-centrifuge in the cold
box (4°C glass-door refrigerator) in MO 412.
6. Follow the nucleic acid purification protocol as written. You will end up with a
lower organic phase in the tube after removal of the upper aqueous phase for
nucleic acid recovery. 8-Hydroxyquinoline is added to the phenol:chloroform
stock solution to tint it yellow and make it visually distinguishable from the clear
upper aqueous phase. Leave this lower phase in the tube and snap the cap
closed. Place to the side of your work area until the purification procedure is
complete.
7. At the end of the purification procedure, take the waste tubes containing the
phenol:chloroform waste to the fume hood, open them and pour out the liquid
into the labeled “Phenol:Chloroform Liquid Waste” bottle. Snap the caps closed
and place the empty tubes containing residual phenol into the bag designated for
phenol-chloroform solid waste along with the pipet tips.
8. Make sure that the start date is on the phenol:chloroform liquid waste bottle, and
record the approximate volume discarded during the session. Close the lid
tightly on the screw-cap waste bottle and keep in the fume hood.
9. When full, both the solid and liquid phenol:chloroform waste are disposed of
according to the NIU EH&S Hazardous Waste Program guidelines. Label the
bottle and bag with the date and amount of Phenol:Chloroform.
http://www.ehs.niu.edu/ehs/chemical/waste.shtml
10. Carefully remove your gloves and place them in the solid phenol:chloroform
waste bag. Clean up the work area with Kimwipes if necessary, and dispose of
them likewise.
11. Remove PPE and wash hands.
#7
WASTE DISPOSAL
We typically generate about 100 ml of Phenol:Chloroform waste every 6 months.
Contact EH&S at 815-753-1610 to schedule a pickup or for questions and additional
guidance.
#8 TRAINING REQUIREMENTS
General Training (check all that apply):
□General Safety & Emergency Preparedness
Chemical Safety for Laboratories
□Radiation Safety
Biosafety training
□Other:________________________
For assistance with this form contact NIU Environmental Health and Safety, 815-753-0404.
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NIU Biological Sciences Standard Operating Procedure
Location Where Records
Maintained:
Montgomery 412 front desk
Laboratory-specific training (check all that apply):
Review of SDS for other chemicals involved in process/experiment
 Review of this SOP
□Other:_________________________
Location Where Records
Maintained:
#9
Montgomery 412 front desk
PRIOR APPROVALS
Prior approvals are required by the following University Committees:
IBC: Recombinant DNA, potential pathogens, human tissue/body fluids
http://www.orc.niu.edu/orc/biosafety/niupolicy.shtml
ORC# S11-0001 and S11-0002
For assistance with this form contact NIU Environmental Health and Safety, 815-753-0404.
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