Problem with drying down polyacrylamide gel - (Jun/24/2008 )
I want to be able to detect multiple bands in a complex sample using autoradiography. So, I am separating 35S[ATP] end-labelled PCR products on a 12% native polyacrylamide gel.
The problem I am having is with drying down the gel, which will subsequently be exposed to film. The gels crack but don't appear to be overdried?
I have tried using 2 different gel driers so I don't think that the vaccum is a problem.
Initally I thought the problem was with the gradient gels I was using, but I am getting the same result with the standard 12% gel.
I have tried drying the gel for 1 hour @ 80 degrees and for 40 mins @ 65 degrees.
A colleague suggested that problem could be that I am not using a gel drying solution before placing the gel on the whatman paper. She gave me a recipe for the solution she uses, but this is used for SDS page. Would this be OK to use? It consists of 25% isopropanol, 10% acetic acid, and 20% glycerol.
Does anyone know of a gel drying solution that can be used for native polyacrylamide DNA gels?
Any suggestions would be appreciated!
Sorry to hear you're having gel drying issues. I had the same problem and after months of pain I finally got a new gel dryer. The only time my gels cracked was when the dryer lost vacuum. I never used a gel drying solution.
I would try to extend your gel drying time - up to 1.5 -2 hours, along with a 15 minute cool down. The thickness of a 12% gel will require a longer drying time, and I would suspect that a gradient gel would have the same problem. I haven't noticed any difference using a gel drying solution as opposed to not using one.
make sure your vacuum pump is operating correctly (pulling a strong enough vacuum).
use glycerol (the solution your colleague gave you is fine, i prefer methanol), it is especially important for high percentage gels. the solution will also shrink the gel so that it won't shrink too much more, and cracking, when drying.
high percentage gels take longer to dry properly.
also, as pointed out by smu2, allow cool down before breaking the vacuum.
how thick is your gel? 0.75mm is ok., 1.5mm will crack.....