sodium acetate in sequencing gel buffers!
kraev at bc.biol.ethz.ch
Sat Mar 8 07:39:09 EST 1997
In article <199703071548.IAA62146 at nestor.NMSU.Edu>, hroychow at NMSU.EDU
(Hiranya Roychowdhury) wrote:
> At 06:16 PM 3/5/97 -0800, zarir wrote:
> >Hi people,
> >i want to know the reason for adding sodium acetate to the lower buffer
> >tank while doing manual sequencing. I know it reduces compressions, by
> >altering the voltage gradient, but how? what is the chemistry involved?
> No, acetate does not do anything for compressions. The acidic anolyte
reduces the electrophoretic mobility of the DNA closer to the anode in
the gel virtually to a crawl. This essentially achieves the effect of a pH
gradient across the gel. The result is that the lower portion of the gel
stacks up the smaller fragments, affording more read per gel without
letting too much of the sequence close to the primer escape. However, it
does take much longer to run.
Actually, the presence of high salt does influence compressions - it
promotes them: as the buffer gradient
formed during electrophoresis also induces the temperature gradient in
the opposite direction. So, compressions
are more likely to reappear in the lower third of the gel, which is
cooler. Such gels are best run with
two thermostatic plates ( a rare commercial device features more than a
single alumina plate backing), otherwise
the upper part can be overheated, and even if it does not crack, the bands
may be blurred. Finally, "much longer"
is an exaggeration: an optimal run is only 50% longer than an equivalent
no-gradient run (typically, 3.5 hours for
a 0.4 mm 50 cm-long gel).
I hope this helps,
Alexander Kraev, PhD
Biochemie III, ETHZ Zurich
e-mail kraev at bc.biol.ethz.ch
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