The
University of Sydney
Title: Casting ethidium bromide agarose gels
SOP Ref No.: #0003
Prepared by: Haydn Allbutt
Reviewed by: Donna Lai
Date: 15/8/07 Thur
Date:
Background
Ethidium bromide (3,8-diamino-5ethyl-6-phenylphenanthridinium bromide; EtBr) is
classified as being low to moderately toxic and is a mutagen and possible carcinogen
and teratogen. Personal protective clothing (gloves, splash resistant eyewear with side
shields, laboratory coat and covered footwear) must be worn at all times when handling
EtBr. Used primarily as a stain for nucleic acids, particularly in agarose gels, you should
avoid cross contamination of materials and solutions to minimize to volume of waste that
needs to be decontaminated and disposed of following the procedure.
Body parts most at risk
Respiratory tract, eyes, hands, skin
Personal protective equipment required






“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
Equipment
Gel casting plates and spacers, 150 ml graduated conical flask, Thermometer, Teflon
stir bar, Stir plate, Pipette tip or toothpick, Tape, Microwave, Glad Wrap
Procedure
A.

B.
Making a 1X TBE solution:
In a 250 ml conical flask, mix 10 ml of 10X TBE with 90 ml of distilled water.
Mixing and Heating the Gel:
1. For a 3% agarose gel, weigh out 3.0 grams of metaphor agarose.
2. Add a white Teflon stir bar to the 1X TBE solution and place on a stir plate.
NB: Ensure that the scale has been calibrated, by verifying an accurate reading with a
300 g weight, prior to use (300mL of distilled H2O should be exactly 300g).
NB: TBE should be cold when adding agarose to avoid clumping.
Bosch Institute
Standard Operating Procedure
© University of Sydney
Page 1 of 3
Procedure (cont.)
3. Set the stir plate to 8 (high) and slowly add the agarose to avoid clumping.
4. Once all agarose is added adjust the stir plate setting to 9 so that the solution
mixes vigorously for 10 to 15 minutes.
NB: The more thoroughly the solution is mixed, the less it will foam when it is heated.
5. Adjust the heat plate temperature to a setting of 6-8 and allow the solution to
come to a slow boil. Maintain a low simmer until all agarose has dissolved
then switch off the heat.
6. 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 EtBr
(10mg/ml stock solution).
NB: The addition of EtBr renders the solution toxic. Solution should be handled with
care. Always use gloves and safety glasses when boiling solutions or handling toxic
solutions.
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 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 Glad Wrap and put the gel in the refrigerator (which is
identified for non-food items).
8. Refrigerate the gel overnight or for at least 6 hours.
NB: 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 ready for re-melting and re-casting.
The flask should be labeled to indicate the date that the gel was cut up and
placed into the refrigerator.
NB: A gel that has been cut up can be stored for up to 20 days.
Bosch Institute
Standard Operating Procedure
© University of Sydney
Page 2 of 3
D.
Re-melting old gels
1. 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 (time will depend on the amount of gel to be heated).
2. While wearing the full face shield, carefully remove the HOT flask from the
microwave with “Hot-hands” insulated holders, or other heat proof insulated
gloves, and place the flask on a stir plate that is turned to the off setting.
3. Verify that the liquid mixture volume is at least 100 ml. If not then add further
hot 3% agarose mixture until the volume is at least 100 ml.
4. Once the gel solution has cooled to 57oC add 5 l of EtBr (10 mg/ml solution)
to the liquid mixture. Pour the agarose solution in to gel casting plates as
described above in section C.
E.
Disposal of waste

See SOP, Disposal of Ethidium Bromide waste (SOP 0004) for details of this
procedure.
Bosch Institute
Standard Operating Procedure
© University of Sydney
Page 3 of 3