These safety training resources, prepared solely for the use of the Regents of the University of California, were provided by a variety
of sources. It is your responsibility to customize the information to match your specific operations. Neither the University of
California nor any of its employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the
accuracy, completeness, or usefulness of any information, or represents that its use would not infringe privately owned rights.
Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does
not necessarily constitute or imply its endorsement, recommendation, or favoring by the University of California. The views and
opinions of authors expressed herein do not necessarily state or reflect those of the University of California, and shall not be used for
advertising or product endorsement purposes.
UC Berkeley
SOP
Standard Operating Procedure
Creating Agarose Gels
(SOP Title)
Principal Investigator:
Lab :
Building:
Contents
Description of Work and Scope of SOP
Required Safety Equipment & Personal Protective Equipment (PPE)
Procedure / Steps Needed To Complete Work Safely
Waste Disposal Procedures
Special Precautions Needed for Acquiring & Transporting Chemicals
Chemicals Used
Mixtures & Ratios
Lab Equipment Needed
Chemical Spill / Release Procedures
Completed By:
Approved By:
Page
2
2
2
3
3
4
4
4
4
Description of Work and Scope of SOP
This SOP addresses the work performed to create an agarose gel. This process begins with mixing TrisAcetate-EDTA (TAE) with distilled water and ends with pouring agarose gel into a gel plate, letting it
solidify, and removing the combs.
Required Safety Equipment & Personal Protective Equipment (PPE)
o
o
o
o
o
o
o
o
“Hot-Hands” Insulated Grippers
Gloves = Heat resistant insulated neoprene gloves
Gloves = Nitrile, at least 4 millimeters thick
Splash Proof Safety Goggles or Safety Glasses
Full Face Shield
Lab Coat
Long Pants
Closed Toe Shoes
Procedures / Steps Needed To Complete Work (Safely)
SPLASH PROOF SAFETY GOGGLES OR SAFETY GLASSES MUST BE WORN AT ALL TIMES WHEN WORKING IN
THE LAB
The following PPE must be used when working with 50X TBE, Ethidium Bromide, or when working with
a 1X TBE solution that contains Ethidium Bromide:
o Nitrile Gloves
o Splash Proof Safety Goggles
o Lab Coat
Note: If any of the chemicals identified above contact your gloves then the gloves should be replaced as
soon as feasible. Ethidium Bromide is a known mutagen. Care should be taken to prevent cross
contamination of Ethidium Bromide. Delineate with tape and label areas where Ethidium Bromide is used
(e.g. “Ethidium Bromide Use Area”).
A.
Making a 50X TAE solution:
a. For 1L 50X TAE combine 242g Tris Base, 57.1 ml Glacial Acetic Acid, and 18.6g
EDTA. Add H2O to 1L mark on Flask.
b. For 1L 1X TAE add 900 ml H2O to 100 ml 50X TAE.
B.
Mixing and Heating the Gel (Example):
1. Add a white Teflon stir bar to the 1X TAE solution.
2. For a 2.0 % Agarose Gel (300 ml), weigh out 6.0 grams of agarose (high melt). Note: Ensure
that the scale has been calibrated, by verifying an accurate reading with the 300 g weight,
prior to use.
3. Prepare to add the 3.0 grams of agarose to the TAE mixture within the flask by placing the
flask on a stir plate and adjusting the stir plate setting to 8.
4. Once the TAE solution (in the flask creates a strong vortex, slowly add the agarose. You
must add the agarose slowly or it will form clumps.
5. Adjust the stir plate setting to 9 so that the solution mixes vigorously for 10 to 15 minutes.
Note: The more thoroughly the solution is mixed, the less it will foam when it is heated.
6. Place the bottle into the laboratory microwave and microwave until the agarose is dissolved.
All of the solids within the solution should have dissolved – if not then heat the solution until
it boils again. Note: observe the agarose in the microwave closely so as not to allow it to boil
over!
2
7.
Using the “Hot-hands” insulated grippers, or while wearing the heat resistant neoprene
insulated gloves remove the bottle from the microwave. Turn the stir plate to a setting of 5,
and allow contents of the flask to stir and cool for a few minutes, until it reaches 58 o C and
then add 5 l of Ethidium Bromide (10mg/ml stock solution). Always use gloves when
handling Ethidium Bromide.
C.
Pouring the Gel
1. Place a gel cast, with taped ends and desired combs on a level countertop.
2. After the gel mixture has cooled to 57o C, pour it into the gel plate.
3. If there are any air bubbles present, pop them or drag them away from the combs to the edge
of the plate with a pipette tip or toothpick.
4. Place a piece of tape on the gel plate to designate that it is a new gel.
5. Let the gel set up and cool for at least 15 minutes on the counter prior to moving. Note: Gels
must be cooled and solid prior to moving them.
6. Wrap the gel with Saran Wrap and put the gel in the refrigerator (which is identified for nonfood items).
7. New gels should be used within 5 days after preparation, otherwise they should be cut up and
put it into a flask. The flask should be labeled to indicate the date that the gel was cut up and
placed into the refrigerator. Note: A gel that has been cut up can be stored for up to 20 days.
D.
Preparing Used Gel
1. Once a gel has been used, cut it up and place it into a graduated 150 ml flask.
2. If the gel is not going to be re-melted the day it is cut up then place Saran Wrap over the flask
containing the cut up gel and store it in the refrigerator that has been identified for nonfood
items.
3. To re-melt a gel, place the uncovered flask containing the gel into the microwave and heat on
high until the gel boils and all of the solid portions of the gel are dissolved.
4. While wearing the full face shield, carefully remove the HOT flask from the microwave with
“Hot-hands” insulated holders, or the insulated neoprene gloves, and place the flask on a stir
plate that is turned to the off setting.
5. Verify that the liquid mixture volume is at least 100 ml. If not then add hot gel mixture from
a filler flask until the volume is at least 100 ml.
6. Once the gel solution has cooled to 57oC add 5 l of Ethidium Bromide (10 mg/ml solution)
to the liquid mixture. Pour the agarose solution as described in the steps found above in
section C.
Waste Disposal Procedures:
Place waste agarose that is contaminated with Ethidium Bromide in the designated gel drying area in the
fume hood.
Emergency Procedures
***Add text here describing the location of the nearest eyewash/shower, how long to rinse, which medical
facility to go to, etc. (consult the EH&S Fact Sheet “Ethidium Bromide and Alternative DNA Stains” as
well as the MSDS for information).
Special Precautions Needed for Acquiring & Transporting Chemicals:
Read and follow guidelines provided in the EH&S Fact Sheet Transporting Chemicals Safely on Campus.
Use common sense when transporting chemicals to minimize the potential for a chemical spill.
Chemicals Used:
**Lab Workers Must Review All Applicable MSDS When Being Trained**
3
The following list of chemicals does not take the place of the required MSDS, which provide information
regarding signs and symptoms of exposure, exposure limits, physical appearance, etc.
Notes: NFPA Ratings and CAS Numbers Can Be Found on most MSDS, and NFPA ratings range from 0 (minimal) to 4 (severe).
Health
Flammability
Reactivity
Ethidium Bromide
CAS
1239-45-8
Starting
Concentration
1%
Acetic Acid
64-19-7
Trade Secret
3
2
2
Tris
77-86-1
Trade Secret
1
1
1
Disodium Dihydrogen
EthyleneediaminetetraAcetatedihydrate
6381-92-6
Trade Secret
N
N
N
Chemical Name
NFPA
Ratings
4
1
1
Other Hazards
Carcinogen, Skin Absorption, etc.
Carcinogen
Note: HMIS or Saf-T-Data ratings may be substituted for NFPA ratings, when NFPA ratings do not exist.
N = Not Determined
Mixtures, and Ratios:
o
o
o
Ethidium Bromide 1% solution.
50X Tris-Acetate-EDTA (TAE) consists of a mixture of Acetic Acid, Tris, and Disodium
Dihydrogen Ethyleneediaminetetra-Acetatedihydrate
Metaphor agarose
Lab Equipment Needed:
o
o
o
o
o
o
o
o
o
Agarose gel plate, ends, and combs
150 ml graduated Erlenmeyer Flask
Thermometer
Teflon stir bar
Stir plate
Pipette tip or toothpick
Tape
Microwave
Saran Wrap
Chemical Spill / Release Procedures:
If a chemical spill of any solution containing Ethidium Bromide occurs then it should be cleaned up while
wearing;
o Nitrile Gloves
o Splash Proof Safety Goggles or Safety Glasses
o Lab Coat
o Long Pants
o Closed Toe Shoes
If the spill is powder, carefully collect the dry powder (because when wet it will stain) and then
quickly wipe up residue with wet paper towels. If the spill is liquid, absorb freestanding liquid
with dry paper towels. Use UV light in a darkened room to locate any remaining solution or
4
stains, then clean area with strong detergent (e.g., tri-sodium phosphate). Use UV light and repeat
decontamination as necessary.
While performing the cleanup, place contaminated absorbent materials in a plastic bag that can be sealed so
that it is liquid tight, for waste pick-up. Label the cleanup materials (e.g., “Laboratory debris
contaminated with ethidium bromide”). Fill out a Materials Packing List to have the waste picked up.
5