UNIVERSITY OF CALIFORNIA, IRVINE
Standard Operating Procedure
Laboratory Specific Processl:
DNA Extraction with Phenol:Chloroform
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Safety-on-Site (SOS) Binder
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_____________________________________________________________________________
Department: Plant Pathology Date when SOP was written: 11-06-12
Date when SOP was approved by the lab supervisor: ___12-16-14________________
Principal Investigator: Douglas Cook
Internal Laboratory Safety Coordinator/Lab Manager: Noelia Carrasquilla-Garcia
Laboratory Phone: 530-754-6489
Emergency Contact:_Noelia Carrasquilla-Garcia 916-7986951_
(Name and Phone Number)
Location(s) covered by this SOP: Robbins Hall, 105,117
(Building/Room Number)
_____________________________________________________________________________
Type of SOP:
Process
Hazardous Chemical
Hazardous Class
Purpose: Chloroform is commonly used in DNA purification procedures in biology and biochemistry
and as a solvent in organic synthesis.
Phenol is only mildly acidic but requires careful handling due to its toxicity and its propensity to cause
severe burns.
Physical & Chemical Properties/Definition of Chemical Group
Potential Hazards/Toxicity
Chloroform is a SELECT CARCINOGEN.
It is harmful if swallowed. Chloroform is irritating to eyes, respiratory system and skin. It poses danger of
serious damage to health by prolonged exposure through inhalation and if swallowed.
Over pressurized containers of chloroform are potentially explosive.
Phenol, also known as carbolic acid, is an organic compound with the chemical formula C6H5OH. It is a
white, crystalline solid at room temperature. It consists of a phenyl (-C6H5) group, bonded to a hydroxyl (OH) group.
Phenol is commonly used in molecular biology techniques such as
from nucleic acids, usually in combination with chloroform. It is a
the central nervous system, liver and kidneys. Phenol is irritating to
SOP Template developed by The UC Center for Laboratory Safety 1
to separate proteins
corrosive that affects
the skin but has a
UNIVERSITY OF CALIFORNIA, IRVINE
local anesthetic effect. Hence, no pain may be felt on initial contact, but severe burns may develop after
sometime. Phenol is rapidly absorbed through the skin so toxic amounts may be absorbed through
relatively small areas.
PHENOL
Synonyms:
CAS#:
Molecular formula:
Molecular weight:
Form:
Hydroxybenzene
Carbolic acid
Benzenol
Phenylic acid
Phenic acid
Phenyl alcohol
108-95-2
C6H6O
94.11 g
white crystalline solid
Phenol is appreciably soluble in water.
CHLOROFORM
CAS# 67-66-3
Colorless, clear liquid
Boiling point 61-62 °C
Formula CHCl3
Potential Hazards/Toxicity
Phenol and its vapors are corrosive and hazardous to the eyes, the skin, and the respiratory tract.
Repeated or prolonged skin contact with phenol may cause dermatitis, or even second and third-degree
burns due to phenol's caustic and defatting properties. Inhalation of phenol vapor may cause lung edema.
The substance may cause harmful effects on the central nervous system and heart, resulting in
dysrhythmia, seizures, and coma. The kidneys may be affected as well. Exposure may result in death and
the effects may be delayed. Long-term or repeated exposure of the substance may have harmful effects
on the liver and kidneys.
Toxic Effects:
Acute Effects:
Chronic Effects:
Phenol is a toxic substance. Phenol is a poison and toxic via ingestion, inhalation
and skin absorption. This chemical is easily absorbed through the skin and
fatalities have been documented from skin absorption through a relatively small
surface area.
Acute phenol intoxication causes shock, collapse, coma, convulsions, cyanosis,
and death. Ingestion of lethal amounts causes severe burns of the mouth and
throat, marked abdominal pain, cyanosis, muscular weakness, collapse, coma,
and death. Tremors, convulsions, and muscle twitching have also occurred.
Contact of the skin with the solid or liquid can produce chemical burns, redness,
edema, tissue necrosis, and gangrene; contact with the eye may result in
irritation, conjunctival swelling, whitened cornea, and blindness.
Chronic phenol poisoning is characterized by vomiting, difficult swallowing,
excessive salivation, diarrhea, anorexia, headache, fainting, vertigo, mental
disturbances, and possibly skin eruptions. Prolonged cutaneous exposure may
result in deposition of dark pigment in the skin.
Carcinogenic Effects:
Not considered carcinogenic
Reproductive Hazard:
May cause reproductive and fetal effects.
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Exposure Limits
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The current OSHA permissible exposure limit (PEL) for phenol is 5 ppm (19 mg/m ) as an 8-hour timeweighted average (TWA) concentration. The OSHA PEL also bears a “Skin” notation, which indicates that
the cutaneous route of exposure (including mucous membranes and eyes) contribute to the overall
exposure.
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The NIOSH established a recommended exposure limit (REL) for phenol of 5 ppm (19 mg/m ) as a TWA
for up to a 10-hour workday and a 40-hour work week. The short-term exposure limit (STEL) for phenol is
15.6 ppm for periods not exceeding 15 minutes. NIOSH also bears the “Skin” notation to phenol.
The American Conference of Governmental Industrial Hygienists (ACGIH) assigned phenol a threshold
3
limit value (TLV) of 5 ppm (19 mg/m ) as a TWA for a normal 8-hour workday and a 40-hour workweek.
The ACGIH also bears a “Skin” notation to phenol.
A worker’s exposure to phenol can be determined by analyzing a urine sample taken at the end of the
shift for total phenol. A 250 mg total phenol per gram creatinine level corresponds to an airborne phenol
exposure at the TLV (5 ppm). Dermal absorption of phenol may contribute to urinary levels found.
Route
Phenol can enter the system by skin absorption/dermal contact, inhalation or ingestion or through eye
contact.
Personal Protective Equipment (PPE)
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Neoprene (double glove), viton or vinyl gloves when handling phenol. Change gloves after
handling phenol. Note: Nitrile gloves are a poor choice when working with phenol.
Safety glasses with side shields or chemical splash goggles shall be worn.
Laboratory coat when working with phenol.
Closed toe shoes are required when working in the laboratory.
If there is a potential for body splash, wear a rubber or neoprene apron.
Notify PI before pursuing respirator use. Lab personnel intending to use/wear a respirator mask
must be trained and fit-tested by EH&S. This is a regulatory requirement. If you think that you
may require respirator use, notify PI and contact EH&S for assistance
(http://www.ehs.uci.edu/programs/ih/respiratory.html)
Engineering Controls
•
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Work with phenol & chloroform in a properly operating and certified chemical fume hood.
Work at least 6” inside the hood, never place your head in the hood, set the sash at the lowest
position possible (if using the horizontal sliding sashes do not open past the green tape mark).
First Aid Procedures
Chemical burns from skin exposures can be decontaminated by washing with polyethylene glycol,
isopropyl alcohol or perhaps even copious amounts of water. Removal of contaminated clothing is
required, as well as immediate hospital treatment for large splashes. This is particularly important if the
phenol is mixed with chloroform (a commonly-used mixture in molecular biology for DNA & RNA
purification from proteins).
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UNIVERSITY OF CALIFORNIA, IRVINE
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In the event of skin contact, immediately wash with soap or mild detergent and large amounts of
water until no evidence of chemical remains (15-20 minutes). Remove contaminated clothing and
shoes immediately if necessary – recommend discarding contaminated clothing. Seek medical
attention.
Olive oil and vegetable oil are effective in removing phenol from skin and retarding absorption.
In case of contact with eyes, immediately flush eyes with copious amounts of water for at least 15
minutes (lifting upper and lower eyelids occasionally) and obtain medical attention.
If inhaled, move to fresh air immediately and seek medical attention.
In the event of ingestion, seek medical attention immediately.
Report all incidents or near misses EH&S and complete the appropriate Incident Forms.
Special Handling and Storage Requirements
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Keep container tightly closed to prevent agent from subliming and entering the atmosphere.
Store away from strong oxidizers such as hydrogen peroxide, chlorine, bromine, acids, and
calcium hypochlorite.
o
Store saturated phenol at 4 C in designated secondary containers.
Wash hands thoroughly after handling (even if gloves were used).
Keep away from heat, sparks, flames, sources of ignition (including empty containers that held
phenol, as the containers will have phenol residue).
Store protected from light and moisture.
Use only in a chemical fume hood.
Transport chemicals in closed containers, in the smallest amounts possible, and use aids such as
carts, chemical transport carriers, etc.
It is highly recommended that all chemicals be stored below eye level so cracking or leaking
containers are immediately visible and there is less potential for chemicals falling onto lab
workers when pulling from shelves.
Spill and Accident Procedure
Note: Do not attempt cleanup if you feel unsure of your ability to do so or if you do not know the proper
procedure to clean up the spill.
Small spills: Use vermiculite or commercially available spill absorbent material. Collect the spilled
material in a clearly labeled hazardous waste container. Place absorbent pillows/pads around and flood
area with water then cover with caustic soda ash to neutralize the residual material.
Large spills: Notify others in room. Evacuate the lab/room immediately. Call 911 and EH&S. Close lab
doors and post warning signs at entrances/exits notifying others regarding the spill. Prevent unnecessary
entry into the spill area. Provide assistance and information to spill responders.
Chemical Spill Dial 911
Spill – Help contaminated or injured persons. Evacuate the spill area. Avoid breathing vapors.
Eliminate sources of ignition if the chemical is flammable. If possible, confine the spill to a small area
using a spill kit or absorbent material. Keep others from entering contaminated area (e.g., use caution
tape, barriers, etc.).
Small (<1 L) – If you have training, you may assist in the clean-up effort. Use appropriate personal
protective equipment and clean-up material for chemical spilled. Double bag spill waste in clear plastic
bags, label and take to the next chemical waste pick-up.
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UNIVERSITY OF CALIFORNIA, IRVINE
Large (>1 L) – Dial 911 (or 310-825-1491 from cell phone) and EH&S for assistance.
Chemical Spill on Body or Clothes – Remove clothing and rinse body thoroughly in emergency
shower for at least 15 minutes. Seek medical attention. Notify supervisor and EH&S immediately.
Chemical Splash Into Eyes – Immediately rinse eyeball and inner surface of eyelid with water for
15 minutes by forcibly holding the eye open. Seek medical attention. Notify supervisor and EH&S
immediately.
Medical Emergency Dial 911
a.
Life Threatening Emergency, Business Hours, After Hours, Weekends and Holidays – Dial
911 CALL 911 if the condition is LIFE THREATENING or REQUIRES IMMEDIATE MEDICAL
ATTENTION.. Note: All serious injuries must be reported to EH&S at x46200 within 8 hours. Complete
online incident report at https://www.ehs.uci.edu/apps/hr/index.jsp
b.
Non-Life Threatening Emergency – Notify your supervisor or faculty staff if condition is not life
threatening.
c.
Needle Stick/puncture Exposure (as applicable to chemical handling procedure) – Wash the
affected area with antiseptic soap and warm water for 15 minutes. For mucous membrane exposure,
flush the affected area for 15 minutes using an eyewash station.
ALL WORK RELATED INJURIES MUST BE REPORTED via the On-line Incident Form
https://www.ehs.uci.edu/apps/hr/index.jsp or call Human Resources, Workers Compensation (949)
824-9152.
Decontamination/Waste Disposal Procedure
Phenol waste is listed as an Extremely Hazardous Waste and hence must be disposed of according to
EH&S guidelines.
Gloves, test tubes, pipette tips, and paper towels, etc. that is contaminated with phenol must be disposed
of as hazardous waste.
Decontamination/Waste Disposal Procedure
Guidance: Describe waste products and disposal procedures for these chemicals. Hazardous Waste
guidelines are located at: http://www.ehs.uci.edu/programs/enviro/
Guidance: Discuss decontamination procedures for work areas, equipment, and glassware. Include
controlled areas (e.g., glove boxes, restricted access hoods, perchloric acid fume hoods, or designated
portions of the laboratory) in your discussion.
Click here to enter text.
General hazardous waste disposal guidelines:
Label Waste
• Hazardous waste labels must be placed on the hazardous waste container upon the start of
accumulation. Labels are available online at www.ehs.uci.edu/programs/enviro/.
Store Waste
• Hazardous waste containers must be kept closed, except when adding waste.
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UNIVERSITY OF CALIFORNIA, IRVINE
Hazardous waste containers must be stored in secondary containment to adequately contain all
of the contents of the container.
• Hazardous waste containers must be inspected weekly for signs of leaks, corrosion, or
deterioration.
Dispose of Waste
• Hazardous waste must be transferred to EH&S for disposal within 6 months of being generated.
• Empty Containers: At no time should full or partially full containers be placed in the trash. For
more information on empty container management visit www.ehs.uci.edu/programs/enviro/.
• Hazardous Waste Disposal:
o Visit www.ehs.uci.edu/programs/enviro/.
o Fill out the “Chemical Waste Collection” form.
o EH&S will pick up your waste within 1-3 days.
• Do not dispose of chemicals by pouring them down the drain or placing them in the trash.
• Do not use fume hoods to evaporate chemicals.
•
Safety Data Sheet (MSDS) Location
MSDS can be found at the laboratory computer located in Room D268 and can be accessed at
http://www.ehs.uci.edu/msds.html.
Hardcopy or electronic copy MSDS from the same manufacturer must be available for Phenol.
Protocol/Procedure
Phenol-chloroform extraction
Purpose: Phenol-chloroform extractions are done when you need to purify DNA from a
solution that also has proteins. The DNA will dissolve in the aqueous layer, and everything
else will go into the non-aqueous layer. If you simply need to concentrate your DNA, or
need to change the buffer, perform only the ethanol precipitation portion.
**Be careful when working with phenol:chloroform!! Wear gloves and use fume hood.
Dispose of waste in appropriate waste containers: tubes and tips in phenol:chloroform
tubes/tips container; liquid waste in phenol:chloroform container.
A. Phenol:chloroform
Add one equal volume of phenol:chloroform to DNA solution. The phenol:chloroform
solution has two layers – be sure to take the bottom layer!!!
Mix gently; spin for 5 minutes at max speed.
Transfer aqueous layer (top layer) to a new tube. Dump the bottom layer into
phenol:chloroform liquid waste container.
Add equal volume of chloroform.
Mix; spin 2 minutes at max speed.
Transfer aqueous (top) layer to a new tube. Dump bottom layer into waste container.
B. Ethanol Precipitation
Add ammonium acetate to 0.3M
Add two and half volumes 100% ethanol
Mix; spin 30 minutes at 4C
Remove supernatant carefully; save to be safe (at least until you quantify to ensure that
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you didn’t lose too much).
Fill tube halfway with 70% ethanol; spin 2 minutes (this is a wash).
Repeat wash.
Carefully pipet out or decant supernatant. There will be a clear pellet on the bottom. It
may be difficult to see.
Dissolve pellet in appropriate amount of TE or desired buffer.
NOTE: Any deviation from this SOP requires written approval from PI.
Documentation of Training (signature of all users is required)
ü
Prior to conducting any work with Phenol, designated personnel must provide training to his/her
laboratory personnel specific to the hazards involved in working with this substance, work area
decontamination, and emergency procedures.
ü
The Principal Investigator must provide his/her laboratory personnel with a copy of this SOP and a
copy of the Phenol MSDS provided by the manufacturer.
ü
The Principal Investigator must ensure that his/her laboratory personnel have attended appropriate
laboratory safety training or refresher training within the last two years.
I have read and understand the content of this SOP:
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