EtBr in agarose gels vs. EtBr staining later
David L. Haviland, Ph.D.
HAVILAND at KIDS.WUSTL.EDU
Mon Mar 6 14:16:27 EST 1995
In <kalch-2302951140480001 at gietz.hgen.umanitoba.ca> kalch at ulam.generes.ca writes:
> In article <D4GKBu.8Ar at midway.uchicago.edu>, rmoldwin at midway.uchicago.edu wrote:
> > If I recall correctly, when I tried it, the EtBr migrated in the opposite
> > direction as the DNA. Also, when you add EtBr to the gel, you can see
> that the
> > bottom of the gel is always darker than the top. I think this is because the
> > EtBr migrates upward. The implication is that low bp bands at the bottom of
> > the gel may not show up under UV light. Am I right about this
> yes there is always the problem that you will have to re-stain anyway but
> for general purposes its easier to have the EtBr in the gel.
> Another trick is to add a little to the bottom buffer chamber on a long
> run to ensure the bottom bands do show up
I know that I'm getting into this thread a bit late but...
There was a Biotechniques article (eons ago!) which basically suggested
adding EtBr to your favorite loading buffer of choice to a concentration of
10ug/ml. That has worked very well for me over the years - so much so that
I've dropped the amount of EtBr that I put into gels. From a 10mg/ml stock
of EtBr, I normally put about 5 ul into a 100-150ml agarose gel, both DNA
and RNA gels. I put about 20ul / liter into my TAE (DNA) running buffer.
For Northern gels, all that goes in is into the samples and 3-4ul into the
gel - nothing in the running buffer.
Doing this has enabled me to photograph virtually any gel shortly after it
has run and rarely does the EtBr become a hinderance.
I'm sure there are others that have had great luck with EtBr (or even
Acridine Orange) staining after running, but for me -- that has been an
endevor in Murphy's law.
Hope this helps,
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