Brigham Young University
SOP
Standard Operating Procedure
Creating 3% MetaphorTM Agarose Gels
(SOP Title)
Principal Investigator: Jeff Maughan
Lab #: 285
Building: Widtsoe
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: Jeff Maughan
Approved By: Jeff Maughan
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Description of Work and Scope of SOP
This SOP addresses the work performed to create an agarose gel. This process begins with mixing TrisBorate-ETDA (TBE) with distilled water and ends with pouring agarose gel into a gel plate, letting it
solidify, and removing the combs. This SOP also addresses re-melting and preparation of used agarose gel.
Required Safety Equipment & Personal Protective Equipment (PPE)
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
Full Face Shield
Lab Coat
Procedures / Steps Needed To Complete Work (Safely)
SPLASH PROOF SAFETY GOGGLES MUST BE WORN AT ALL TIMES WHEN WORKING IN THE LAB
The following PPE must be used when working with 10X 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. According to the MSDS, TBE can be absorbed through the skin and Ethidium Bromide is
a known mutagen. Care should be taken to prevent cross contamination of Ethidium Bromide.
A.
Making a 1X TBE solution:
1. In a 250 ml Erlenmeyer flask, mix 10 ml of 10X TBE with 90 ml of distilled water.
a. It is preferable that the solution be chilled before use; otherwise the MetaPhorTM
agarose will clump when it is added.
B.
Mixing and Heating the Gel:
1. Add a white Teflon stir bar to the 1X TBE solution.
2. For a 3% MetaPhorTM gel, weigh out 3.0 grams of metaphor agarose. 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 MetaPhorTM agarose to the TBE mixture within the flask by
placing the flask on a stir plate and adjusting the stir plate setting to 8.
4. Once the TBE solution in the flask creates a strong vortex, slowly add the MetaPhorTM
agarose. You must add the MetaPhorTM 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. Adjust the heat plate temperature to a setting of 6-8 and allow the solution to come to a slow
boil. All of the solids within the solution should have dissolved – if not then heat the solution
until it boils again.
7. Reduce the heat plate setting to 0 or move the flask from the heat plate to a stir plate (without
heat) using the “Hot-hands” insulated grippers, or while wearing the heat resistant neoprene
insulated gloves. Note: Always have stir plates turned to the off setting prior to placing
solutions on them to avoid “flare-ups”.
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8.
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 58o 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 plate, with the ends installed, on a level countertop.
2. After the gel mixture has cooled to 57o C, pour it into the gel plate.
3. Place the gel combs in the appropriate areas of the plate right away.
4. 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.
5. Place a piece of tape on the gel plate to designate that it is a new gel.
6. 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.
7. Wrap the gel with Saran Wrap and put the gel in the refrigerator (which is identified for nonfood items).
8. Refrigerate the gel overnight or for at least 6 hours. Note: the wells will collapse when the
combs are pulled out if the gel is not allowed to chill in the refrigerator for at least 6 hours..
9. 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.
i. A filler gel mixture consists of re-melted 3% MetaPhorTM gels designated as
filler only.
6. Once the gel solutions 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.
i. If you will not pour the mixture until the next day, cover the flask with
saran wrap, and place the agarose solution in an incubator oven or water
bath that is set at 60o C.
ii. Remove the flask from the incubator and follow the steps outlined above in
sections B9 through C10 for pouring.
Waste Disposal Procedures:
Place waste agarose that is contaminated with Ethidium Bromide, in a sealable, liquid-tight, labeled waste
container, and contact Chemicals Management (422-6156) to have them collect the chemical waste.
Special Precautions Needed for Acquiring & Transporting Chemicals:
Standard precautions should be implemented when transporting the chemicals used for this lab work.
Standard precautions require that chemicals be transported in a manner that minimizes the potential for a
chemical release.
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Chemicals Used:
**Lab Workers Must Review All Applicable MSDS When Being Trained**
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%
Boric Acid
10043-35-3
Trade Secret
1
0
0
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.
10X Tris-Borate-EDTA (TBE) consists of a mixture of Boric 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 TBE or Ethidium Bromide occurs then they can be cleaned up while wearing;
o Nitrile Gloves
o Splash Proof Safety Goggles
o Lab Coat
Paper towels can be utilized to absorb the spilled chemicals.
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If the spill involves Ethidium Bromide then a UV lamp should be utilized to check for contamination and to
verify that the Ethidium Bromide has been cleaned up thoroughly. Once the gross quantity of spilled
chemicals has been cleaned up then moist paper towels should be used to wipe the area clean.
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. Contact Chemicals Management (422-6156) to have the waste
picked up.
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