DNA sequencing gels

bugg at mbcf.stjude.org bugg at mbcf.stjude.org
Wed Jul 21 13:36:03 EST 1993


In article <01H0QD3W2F5K94DP4H at Butler.EDU>, KARN at Butler.EDU writes:
> Dear Sequencing Netters:
> 	I have relatively little experience running DNA sequencing gels and
> am having problems with samples spreading across other lanes when I am loading
> them.  I use a BRL S2 gel of the standard 0.4 mm thickness and the usual
> concentrations of gel components and urea.  It seems that I have this problem
> more often (but by no means soley) when I pour the gel the night before and
> run it the next day (I have been led to believe that this practice is accep-
> table if one takes measures to keep the gel from drying out while sitting
> overnight).  The spreading problem does not seem to be due to the shark's
> tooth comb penetrating the gel to any particular degree.  Sometimes I get
> very good loading (i.e. no spreading across lanes) and sometimes gross cross-
> lane contamination - and I can see no pattern to why it works well sometimes
> and not others.  I would be most grateful for tips from you oldtimers in the
> sequencing game.
> Bob Karn
> KARN at BUTLERU.edu

It is interesting that the problem is worse if you pour the gel the night
before.  This often results in more little pieces of acrylamide getting stuck
under the shark's teeth of the comb.  If they do get stuck there, what you end
up with is a small piece of acrylamide that "connects" two wells together by
passing under the tooth which is supposed to separate them.  If this is the
problem, make sure you rinse the big well very well before you put the comb
down into the gel.  Also, I make a little tool to remove these pieces by
cutting a diagonal across the edge of a piece of pH "paper", which is really
made of thin plastic.  You could also use a piece of one of the thinner spacers
which you don't need for sequencing.  The idea is to have something which is
not as thick as the spacers between your plates which you then use to "scoop"
out anything that remains in the well before you put the comb in.  I always
have to do this when the gel has sat overnight, even though I put buffer in the
well area and cover with Saran wrap before leaving the gel.
The previous response you got concerning the clamps already discussed the other
idea I had to suggest.  It is very important that the well end of the gel
should be clamped because otherwise the gap between the plates can be larger
than the thickness of the comb and DNA solution can slip behind the comb.
Good luck!
Barbara Bugg
Molecular Pharmacology
St. Jude Children's Research Hospital
Memphis, TN  USA



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