PAGE gel polymerization

J. Martinez-Irujo jjmirujo at unav.es
Fri Nov 5 04:15:47 EST 1999


Rob Jordan 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

Making a 12% acrylamide gel should not be a problem... In my opinion,
your recipe it's right. It sounds as a problem in the polymerization
reaction to me. We have had similar problems with 4-5% acrylamide gels
(adding more APS was the solution). Try to increase its concentration
(though 70 ul should work). Test your polymerization variables: stock of
APS (is daily prepared?), TEMED, proper degassing, temperature, time of
polymerization...), and tell us what the problem was when you find the
answer... Good look


Juan José Martínez Irujo

Departamento de Bioquimica
Universidad de Navarra
Spain






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