Making an Agarose Gel
Materials
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A microwave safe beaker or flask
Microwave oven
1x or 0.5x TAE/TBE Buffer
Agarose LE (Agarose Powder, GoldBio Catalog # A-201 or Agarose Tablets, GoldBio Catalog # A-205)
A balance
Plastic Wrap
Thick gloves or potholders
Gel casting device
Method
Melting agarose using a Microwave Oven
1. Using a beaker or flask that is 2-4x the volume you are making, add 1x or 0.5x TAE or TBE buffer to
the necessary volume. Use room temp buffer if possible.
1x Tris-Acetate-EDTA (TAE)
40mM Tris (pH 7.6), 20mM Acetic Acid, and 1mM EDTA.
50x Stock (1 liter) - Dissolve in 600 ml distilled water:
242.28 g Tris base [Tris Buffer, GoldBio Catalog # T-400, (MW = 121.14 g/mol)]
57.1 ml glacial acetic acid
100 ml 0.5M EDTA (pH 8.0) [EDTA Disodium, GoldBio Catalog # E-210, (MW = 372.24
g/mol)]
Fill to a final volume of 1 liter with distilled water.
1x Tris-Boric Acid-EDTA (TBE)
89mM Tris (pH 7.6), 89mM Boric Acid, 2mM EDTA
10x Stock (1 liter) - Dissolve in 600 ml distilled water:
107.81 g Tris base [Tris Buffer, GoldBio Catalog # T-400, (MW = 121.14 g/mol)]
55.03 g boric acid [Boric Acid, GoldBio Catalog # B-031, (MW = 61.83 g/mol)]
40 ml 0.5M EDTA (pH 8.0) [EDTA Disodium, GoldBio Catalog # E-210, (MW = 372.24
g/mol)]
Fill to a final volume of 1 liter with distilled water.
Note: TAE provides faster electrophoretic migration of linear DNA and better resolution of supercoiled
DNA. TBE buffers have a stronger buffering capacity for longer or higher voltage electrophoresis.
2. Weigh out the agarose and add it to the flask. To hydrate, swirl the beaker and suspend the agarose
in solution. Alternatively, you can use a stir bar and stirring plate to rapidly mix the solution, but
remember to remove the stir bar before microwaving.
a. Let the agarose hydrate a minute or two before proceeding, this allows for a quicker dissolution
and can reduce foaming. Let higher percentage gels (>1.5%) hydrate longer than lower
percentage gels.
Gold Biotechnology
Web: www.goldbio.com
St. Louis, MO
Ph: (314) 890-8778
email: [email protected]
Optimal Gel Concentration for Best DNA Separation
TAE/TBE
0.5
0.75
1.0
1.2
1.5
2.0
DNA Size Resolution (bp)
1,000 - 25,000
800 - 12,000
500 - 10,000
400 - 7,500
200 - 3,000
50 - 1,500
3. Cover the mouth of the beaker with plastic wrap, and make a small hole in the top to allow the
solution to vent.
4. Weight the beaker and write down the starting weight.
5. Heat the beaker in the microwave for 1 minute or until the solution begins to boil.
6. Remove the beaker from the microwave and very gently swirl.
WARNING: THE MICROWAVED SOLUTION CAN BECOME SUPERHEATED AND FOAM OVER
QUICKLY WHEN AGITATED. USE CAUTION AND ALWAYS WEAR APPROPRIATE PROTECTION.
7. If solid agarose or gel pieces remain, return the flask to the microwave and continue heating in 30
second intervals until all product is in solution. This may take up to a few minutes depending on the
strength of your microwave and the gel concentration you are making.
8. Once the gel is fully melted, reweigh the solution and add dH20 to the beaker to reach the starting
weight. Mix thoroughly.
9. Cool the solution to around 60°C, then cast the gel following the instructions provided for your
casting apparatus.
Gold Biotechnology
Web: www.goldbio.com
St. Louis, MO
Ph: (314) 890-8778
email: [email protected]