deaerate or not deaerate

Dr. Peter Gegenheimer PGegen at UKans.nolospamare.edu
Fri Aug 21 18:00:58 EST 1998


On Thu, 20 Aug 1998 14:20:03, "Zbigniew Rudzki" <mprudzki at cyf-kr.edu.pl> wrote:

> Does degasing of polyacrylamide belong to the group of totally magic and
> irrational procedures contaminating molecular biology ?
> Today, forced by the technical reasons, I tried for the first time to skip
> it and I added twice as much TEMED as usually (20 uL per 70 mL).  It
> resulted in an almost instant catastrophic polymerization :)
>
> So what is your opinion on degasing, after all rather troublesome a
> procedure ?

Polymerization of acrylamide with Ammonium persulfate and TEMED is inhibited by
oxygen. Polymerization is faster and, most importantly, more uniform, in the
absence of oxygen. That's why, when you make a gel with a stacking layer, you
cover the separating gel with water or hexanol to exclude air.

The reason that deaeration usually doesn't have much effect is that gels are
most often prepared from liquid stocks which have been sitting around for long
enough that most of the dissolved air is lost. A freshly-mixed stock of
room-temperature acrylamide, or acrylamide plus buffer and urea, will have a
lot of dissolved air; after sitting a week or two at 4C the dissolved air will
have reached equilibrium at (..what I have been told is...) a lower
concentration.

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