deaerate or not deaerate

Sam Michaelson sam.michaelson at dsto.defence.gov.au.ANTISPAM
Sun Aug 23 21:09:08 EST 1998


Dr. Peter Gegenheimer wrote:

> 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 ?

Degassing of acrylamide solutions was practised by the Devoted Acolytes
of the Great God Gel Pour in the lab where I was a post-grad, using
BioRad Mini-Protean apparatuses (apparati?).  I was told, by supervisor,
more senior labmates and several protocol books that polymerisation of
acrylamide by APS and TEMED is inhibited by oxygen and that degassing
was 'essential'.  Perhaps if you were pouring large slab gels, the extra
time taken by a non-degassed acrylamide solution would be significant,
but I have never poured large gels and so cannot say whether this is the
case.

However,  I found that I usually had a bit more time to pour the gel,
got a nice flat gel surface on top of the resolving gel, got less
shrinkage of the stacking gel around the wells, and got more consistent
results when I omitted the degassing procedure (which, as far as I could
make out, only ever served to take up more time, and also to get little
bits of rubber from the stoppers into the gel mix).  The Cult of the
Omitters of the Degassing Step acquired quite a few followers - for
reasons I could never fathom, a couple of co-workers took this Very
Seriously Indeed and became quite upset about it.  (Fortunately, my
supervisor was an ecumenical sort of guy, and never got worked up about
protocol heresy, as long as it worked and his students got their work
done.)

Sam Michaelson


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