Acrylamide Gel setting too quickly -- IDEAS??
wolffie at u.washington.edu
Mon Jun 10 12:00:55 EST 1996
In article <4oqlfi$8iu at dfw-ixnews9.ix.netcom.com>, macnevin at ix.netcom.com
(Brian MacNevin) wrote:
> Well, when I mix up my acrylamide gel solution and begin pipetting it
> between the glass plates for electrophoresis, I have found that it
> starts to set before I can get it evenly dispersed in the plates.
> My first thought is that this might be due to the temp of the
> solution. It seems to me that I don't have this problem if the
> solution is allowed to cool a little before adding the final solution.
> I was wonderin if anyone else has had these problems and if it really
> is due to the temp of the solution?
Bio-Rad has a nice technical bulletin on PAGE that we've found helpful.
In our lab, the degassing vacuum was found to be a MAJOR variable, as was
the use of a magnetic stirrer to aid degassing. I'd advise against
cooling the reagents -- this increases O2 solubility -- just degas the
hell out it for 15' with a stirrer (warm it up SLIGHTLY first). Keep a
strict log of every gel you pour; this helps to spot problems and
maintain quality assurance.
I think I cut the "standard" (please don't laugh) catalyst recipe to about
65% (you don't want it to set too fast, or the polymer chains will be too
short). For a denaturing sequencing gel, I make up a 67 mL batch either
6% or 8%, and use 44 uL TEMED and 220 uL fresh 10% APS.
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