z-suldan at ski.mskcc.nospam.org
Wed Sep 16 07:36:12 EST 1998
In article <jmmana-1609980830560001 at 184.108.40.206>, 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?
> Magie Chokloikaew
> University Of Kentucky
> Dept. Otolaryngology/Molecular Genetic Research
> 800 Rose Street
> Lexington, KY 40536-0084
> 606-2575096 fax
> mchokl at pop.uky.edu
Do you mean through the sides of the wells or over the top of the wells
and down into the next? I've found the best solution for a loose
sharktooth comb is just clamps and weight. I put two clamps on either side
of the comb at the top of the gel and then one or two clamps coming down
right over the comb (This is bit harder because one plate is of course
shorter than the other). I then lay the gel almost flat on the bench and
place two heavy bottles of liquid on top of the plates.
If you're finding leaking over the top of the well, just load less. I
don't load more than 3uL of sequencing sample into our shartooth wells.
One other possibility, have you measured the spacers? We routinely mix up
our 0.5mm and 0.4mm spacers. If I remember, we got a little plastic device
from BioRad (??) that'll measure the thickness of spacers, combs, etc.
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