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The Laboratory of Steve Hahn at the Fred Hutchinson Cancer Research Center
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1. Preparation of the Acrylamide Urea Gel.
For a 10% Acrylamide Urea Gel, make the following solution:
10 ml 40%:2% Acrylamide:Bisacrylamide
3.5 ml 10X TBE Buffer
15 g Urea
10 ml ddH2O.
For a 15% Acrylamide Gel, make the following solution:
13.1 ml 40%:2% Acrylamide:Bisacrylamide
3.5 ml 10X TBE Buffer
15 g Urea
7 ml ddH2O.
2. Microwave the solution in a side-arm Erlenmeyer flask for about 10 sec. Swirl the flask to dissolve all the solids in the solution.
3. Degas by hooking up the flask to a vacuum line and applying the vacuum for 1 to 2 min.
4. Next prepare the gel casting plates. Siliconize only the plate that has the ears. With the proper spacers in between the plates at the perimeter (on the bottom three sides only), clamp the two plates together (see Hint #2).
5. To the Acrylamide solution, add 0.3 ml of 10% Ammonium Persulfate and then add 35 μl of TEMED.
6. Pour the Acrylamide solution between the casting plates and add a comb to the top of the gel between the ears of the front glass plate.
7. Wait at least 20 min for the Acrylamide to fully polymerize before attempting to remove the comb.
8. The gel can be stored overnight at room temperature if the top of the gel is wetted with water and the whole gel is wrapped tightly in plastic wrap.
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| 10% (w/v) Ammonium Persulfate |
| Make up fresh just before use
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| TBE Buffer (10X) |
| 2 mM EDTA, pH 8.0
89 mM Tris
89 mM Boric Acid
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| 40%:2% (w/v) Acrylamide:Bisacrylamide |
| CAUTION! see Hint #1
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Bisacrylamide
Tris
EDTA
TEMED
Ammonium Persulfate
Boric Acid
Acrylamide
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Adding a little petroleum jelly on the edge of the plates where the pieces of the separate spacers come together will help prevent leakage while pouring the gel.
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