When I casting Urea PAGE gel, I can see the gel shrink in bottom and no wells formed but gel solidified. Does anyone know a possible reason or how to minimize this?
Oxygen inhibits polymerization. Try degassing your gel solution before pouring the gel. Use a fresh solution of ammonium persulfate.
Does your gel solidify unevenly throughout the plate (patchy) or just the top and bottom? In the first instance, try using a little less TEMED and immediately homogenize the solution without introducing bubbles, allowing solidification more time than you currently allow. 2nd instance, you need to review how you are setting up the plates. Taping up the edges properly and placing the comb early as possible helps. As mentioned before, when in doubt, fresh APS.
Along with the other two good suggestions I'd recommend using only fresh urea, as it is not a very stable compound, especially in solution.
Look closely whether the comb touches the gel mixture or whether there are bubbles between the two. In the latter case, air oxygen may prevent polymerisation, leading to malformed wells. I replaced the manufacturers combs with home-made to solve the problem.
I have many suggestions.
1. Try to add more gel after pouring of your gel in to casting tray and after placing comb in to the plate.
2. Check any linkage
3. Use recommended concentrations of all the chemicals (try to use new)
4. Clam should be on the bottom half of the plate not just near the comb.
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