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

Greetings:

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,
David




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