PAGE gel polymerization

Bonehammer g5003408 at uts.univ.trieste.it
Thu Nov 11 13:01:23 EST 1999


In article <7vsqoc$i70$1 at nntp4.u.washington.edu>,
  "Rob Jordan" <rjordan at u.washington.edu> wrote:
> Recently I've started using my own cast TBE page gels instead of
precast.
> I'm using an apparatus from Owl to make 8x10cm, 0.8mm thick gels. My
problem
> is that the wells aren't formed very well. I'm using a 10 place
rectangular
> tooth comb to form wells, and the problem is that when I remove the
comb the
> wells aren't nice and straight and rectangular. The sides of the wells
> especially are irregular.
>
> I'm thinking that the problem is in the polymerization. Here is the
recipe
> that I've been using for 10 ml of gel solution (12%):
>
> 40% acrylamide, 3ml
> 5X TBE, 2ml
> H20, 5ml
> 10% APS, 70ul
> TEMED, 4ul
>
> Now that I think about it does it work better to put the comb in
teeth up
> for polymerization, then turn it over and use the space between the
teeth to
> form the actual wells? Hmmm...that's probably the way you're supposed
to do
> it...
>
> Thanks-
> Rob
>
>
Here is my $0.99 worth contribution: you probably already know 99,9% of
it so please be patient...
Wash and rinse every single piece of glassware. If you are going to use
urea, use a very pure grade, keep the beaker in lukewarm water and stir
with a sprinkle of Amberlite. Degas by vacuum pump and place the
acrylamide container on ice. Add APS, shake a little, add TEMED, shake
again. Inject the stuff between the glasses, insert the comb slowly and
keeping it slightly tilted towards the "eared" glass. Clamp the comb
and don't let any living soul near the gel for the next hour. Check
polymeryzation progress by looking at the unused acrylamide, not at the
gel. When you extract the comb, move it a little before pulling it out.
This helps separation between the gel and the plastic.
BTW do you use silane on the glasses? Maybe the wells get distorted
because the gel is not bound strongly enough to the "not-eared" glass.
The stuff I use to do this is:
-dimethil-dichlorosilane or "non-binding" on the "eared" glass
-methacryloxypropyltri[methil?]silane or "binding" on the other in a
solution made by:
-5 ml EtOH/binding silane (50 ml/150 ul)
-75 ul acetic acid 10%

Hope this helps.
Anyway, if you fix the problem and have some spare time, drop me a line
to know what you did.

Chiara Martellossi
--
"No se Pol"

Coffee Genetist from Viva La' e Po' Bon City


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