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1. Carefully clean the plates with soap, water, 70% ethanol, and
last 100% ethanol. Wipe clean with Kimwipes.
2. Let the plates air-dry.
3. Assemble plates and spacers (clean sides facing each other).
4. Clamp with binder clippers.
5. Prepare 8% polyacrylamide/urea solution:
| Urea |
40 g |
| 30% polyacrylamide |
21.3 ml |
| TBE (10X) |
8 ml |
| dH2O |
to 80 ml |
6. Dissolve urea by stirring. Warming to 50 °C may be needed to completely
dissolve urea.
7. For best results, filter through 0.2 micron filter and de-gas for 5-10
minutes.
8. Immediately before pouring gel, add 0.8 ml of 10% ammonium persulfate
and 50 ml of TEMED.
9. Pour the gel with a 25 ml pippet or syringe.
10. Starting from one side with a continuous stream to avoid trapping
of air bubbles.
11. Air bubbles may be removed with gentle tapping or a thin spacer.
12. Insert comb. For the shark teeth comb, insert with the teeth facing
upward (invert after the gel has polymerized).
13. Clamp the top with binder clippers.
14. After the gel has polymerized, remove the bottom spacer and
assemble on the running apparatus.
15. Fill the top and bottom tanks with 1X TBE.
16. Remove or invert comb.
17. Clear the wells of precipitated urea with a needle connected to a syringe.
Blow with the running buffer.
18. Boil the sample with loading buffer.
19. Pre-run the gel.
20. Load the samples. Run with a constant power (e.g., 65 W).
See also Effective range of separation of DNAs in PAGE/Urea and
Dye migration in agarose and polyacrylamide gels.
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