ELECTROPHERISIS

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Wed Sep 16 18:06:28 EST 1998


In article <jmmana-1609980830560001 at 128.163.85.156>, jmmana at pop.uky.edu
(jose manaligod) wrote:

> We are running 6% Acrylamide gels using .4mm spacers and combs.  We are
> having a lot of problems with our samples leaking out of the wells into
> the one beside it.  After our PCR we use 8ul of the PCR sample and 3ul of
> dye.  We greese the comb with vaccum grease.  Has anyone had this problem,
> if so do you know how to prevent leaking?

I'm not sure if the vacuum grease is the problem.  You may want to
consider using clean ungreased combs instead.

If the gel is poorly formed at the top then consider degassing the
solution or adding riboflavin to allow better polymerisation in the
presence of oxygen.

If you are seeing "creeping" of samples between wells then consider
rinsing the top of the gel before loading and/or check the salt/organic
concentration of your samples.  Add one tenth of a volume of x10 TBE
(or your running buffer) to stop levitation of your samples from the
wells.

If the top of the gel is not correctly attached to the glass plate
then clean the plates scrupulously.  Maybe leave off the silanisation
in this region.  Careful when handling the gel sandwich - if you clamp
too firmly at the bottom you can cause the plates to separate at the
top.

     Good luck,
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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