Help: PAGE sequencing gels
Lori Sadler Sullivan
gsbs047 at utsph.uth.tmc.edu
Tue Feb 21 11:09:13 EST 1995
In article <wolffie-2002951800380001 at 184.108.40.206>,
wolffie at u.washington.edu (John Wolff) wrote:
> Some of my 6% polyacrylamide gels seem to mysteriously swell while I'm
> warming them up: the gel seems to get squeezed upwards, out of the
> plates, and the wells get unusably distorted, and the gel ruined.
> I don't think I'm warming them too fast; we used to equilibrate them at
> 75 watts, and this has happened at 60 watts.
> We recently started using dehydrated TBE buffer instead of pre-mixed
> stuff, and this does smell of some kind of osmotic problem.
> I've also been experimenting with the polymerization recipe.
> I often pour the gels 1-to-4 days before they are used, and this has
> happened both with older 1-day-old gels. I don't think that's the
> trouble. I wrap the comb end with Saran wrap and leave a paper towel
> moistened with 1X TBE adjacent to the end to retard drying-out.
> Must such gels be stored perfectly flat? Sometimes I leave them inclined
> upwards at a very shallow angle (<5°).
> Could it be thermal warping of the glass plates?
I too have had this happen a number of times over the years and the only
correlation I've ever been able to make is that if you have recently
siliconzed the plates, it is more likely to happen (as if the plates are
TOO slippery and the gel is sliding out). I routinely let my gels sit for
a day, at an angle, and cover the tops with Saran wrap.
As for thermal warping of the plates, that must happen sometimes because
I once (and only once) prewarmed a gel after taking the comb completely
out and was then unable to get the comb back IN when it was time to load it.
I tried everything I could think of to get the comb back in but the plates
had squeezed shut (but the gel stayed in place!). In the end I had
to start over. And yet most other times, the plates warm up and the combs get
LOOSE. If someone can explain this to me...
More information about the Methods