Gel electrophoresis: samples running towards the middle?
Dr. Hiranya S. Roychowdhury
hroychow at nmsu.edu
Thu Dec 18 16:10:12 EST 2003
I haven't had such luck with even when I pourted the stacking gel the night
And, I did mention that constant voltage is superior to constant current so far
as "crispy" separation is concerned. For me, mini gels (0.75mm) run in 45 min
at 150V :-)
Quoting Kyle Legate <legatek at hotmail.com>:
> "Dr. Hiranya S. Roychowdhury" wrote:
> I agree with all of these points, with two comments:
> > 5. The Stacking layer (low pH) should not be poured until one is
> > ready with the samples and is absolutely sure that the electrophorsis
> > will begin within about an hour!
> I have routinely poured 30 gels at a time in a multicaster and stored them
> upright, combs intact, in a sealable plastic container with a thin layer of
> water at the bottom, in the coldroom for 2-3 weeks at a time with no
> noticeable decrease in quality.
> > 8. Although most protocols talk about constant current, constant
> > voltage works much better in producing "pretty" pictures. The
> > decreasing PD under constant current causes the low Mr polypeptides
> > to show up diffused. Also, the voltage/current should be kept at the
> > optimum.
> I run Biorad mini gels (~5cm separating length) at 200 volts constant
> voltage. It takes around 45 minutes for the dye to reach the bottom, and I
> have never had a problem with fuzzy bands above, say, 6 kDa.
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
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