SOP DNA and RNA extraction Starr Lab
Title or Type of Procedure: Extraction of DNA and RNA from human and mouse
cells or tissues
P. I. Timothy K. Starr
Lab Location: 12-175 MoosT
Original Issue Date: 4/12/11
Prepared By:
Revision Dates:
5/21/11, 3/4/14
Timothy K. Starr Approval Signature:
(if required by lab supervisor)
Procedural Methods and Materials:
General Safety Procedures:
Wear Personal Protective Equipment (PPE): sterile gloves, eye goggles and lab coat.
All work involving trizol, phenol, or chloroform must be performed in a Fume Hood.
Procedural Methods and Materials:
The following is a list of general steps involved in the RNA and DNA isolation process.
Each step will have important safety information denoted for that aspect of the procedure.
1. General Precautions:
a. Trizol is toxic in contact with skin or swallowed. Causes burns with direct
contact (phenol component). If contact with skin or mucous membranes
occurs, wash immediately with copious amounts of water and detergent.
Seek medical attention if surface area or depth of involvement is too
extensive for treatment with water and detergent i.e. not improving with
this intervention.
b. Use of disposable tubes made of clear polypropylene is recommended
when working with less than 2-ml volumes of TRIzol reagent.
c. For larger volumes, use glass or polypropylene tubes, and test to be sure
that tubes can withstand 12,000xg with TRIzol reagent and chloroform.
Do not use tubes that crack or leak.
d. Carefully equilibrate the weights of the tubes prior to centrifugation.
e. Glass tubes must be sealed with parafilm and topped with a layer of foil,
and polypropylene tubes must be capped prior to centrifugation.
f. Disposable chucks should be laid down where work with tissue or it’s
extracted components occurs. Chucks should be disposed of in Biohazard
bins once extraction is complete
2. Homogenization:
a. Homogenization should be carried out under the fume hood due to phenol
component
b. Fume hood should act as designated area for working with Biosafety level
2 agents (HBV, HCV, other blood born pathogens). This is to be assumed
whenever working with human blood or tissue.
c. Personal protective equipment should be worn such as lab coat, gloves,
and goggles.
3. Phase Separation:
d. This step should also be performed in the fume hood as it entails open
exposure to both phenol and chloroform. Once aqueous phase has been
separated, remainder of protocol can take place at a designated bench
(your bench).
e. This step involves centrifugation. Please see general precautions about
centrifugation.
4. RNA Precipitation
a. Please see general precautions about centrifugation
5. RNA Wash
a. Please see general precautions about centrifugation
6. Redissolving the RNA
7. DNA Precipitation and Wash
a. This occurs using the interphase from the original phenol/chloroform
extraction. Once the interphase is removed, the remaining steps can occur
at the bench
b. Please see general precautions about centrifugation
8. Redissolving the DNA
Hazard Identification and Risk of Exposure to the Hazards:
Waste Generated and Disposal Methods
Please see attachment
Spill and Accident Response Procedures
Please see attachment
Hazard Identification and Risk of Exposure to the Hazards:
The two main hazards for this protocol are exposure to skin or mucous membranes by
material from tissue or cells or exposure to toxic reagents.
Exposure Controls Specific to Above Risk of Exposure:
PPE: Lab Coat, safety goggles, gloves, and masks.
As stated previously, wash with water and detergent if exposed to tizol, phenol or
chloroform and seek medical attention if warranted. Bring MSDS to area where seeking
medical attention. If you suspect that a natural barrier may be broken and internalization
of tissue components may have occurred, seek medical attention. Although the use of
sharps are not specifically called for in this protocol, if one uses them in the
homogenization step, do not recap needles and dispose of all sharps in designated sharp
containers.
Waste Generated and Disposal Methods:
Liquid waste will be collected in a glass jug labeled as hazardous waste and listing the
chemicals. The glass jug will be stored in the fume head with a cap inside a
secondary container. When glass jug is 90% full, jug will be disposed of as
hazardous waste using DEHS disposal service.
Solid waste that has come into contact with trizol, phenol or chloroform will be placed in
a bag inside a sealed bucket and labeled as hazardous waste. When bucket is 90%
full it will be sealed with taped and disposed of as hazardous waste using DEHS
disposal service.
Sharps containers will be sealed when ¾ full and placed in designated waste area.
Refer to the Biological Waste Disposal procedures posted on the Tissue Culture room
door for more information
Spill Response Procedures:
Cover any spills with paper towels, and place towels in bucket labeled as hazardous
waste. If exposed to trizol, phenol, or chloroform immediately rinse with copious
amounts of water and seek medical attention at Boynton Health Services, or Fairview
Emergency room if after normal business hours.
Accident Response Procedures:
If Incident Results in a Hazard Exposure ( i.e. face or eye splash, cut or puncture with
sharps, contact with non-intact skin):
• Encourage needle sticks and cuts to bleed, gently wash with soap and water for 5
minutes; flush splashes to the nose, mouth, or skin with water; and flush eyes at the
nearest eyewash station with clean water for 15 minutes.
• Call 911 or seek medical attention.
- For urgent care employees may go to HealthPartners Occupational and
Environmental Medicine (M/F day time or Urgent Care after hours), or UMMCFairview Hospital (24 hrs). You may seek medical attention at the closest available
medical facility or your own healthcare provider.
- Follow-up must be done by HealthPartners Occupational and Environmental
Medicine.
• Report the incident to your supervisor as soon as possible, fill out the appropriate
documentation.
- Employee First Report of Injury
- Supervisor Incident Investigation Report
• Send Incident Report Form to the IBC if exposure has occurred during work on an
IBC protocol.
• Report all biohazard exposures to the Office of Occupational Health and Safety (612626-5008) or uohs@umn.edu.
Note: It is important to fill out all of the appropriate documents to be eligible to collect
workers compensation should any complications from the hazardous exposure arise in
the future.
Notes: (special record keeping such as inventories for toxins, reporting, training, etc.
that may be required)
References:
For further information view the UMN DEHS website containing Bio Basic Fact Sheets
at http://www.dehs.umn.edu/bio_basicfacts.htm.
For general information on Biosafety, access the Biosafety in Microbiological and
Biomedical Laboratories (BMBL) 5th Edition from the CDC at BMBL
http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
For Material Safety Data Sheets access the Public Health agency of Canada website
MSDS http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php.