RNA electrophoresis!!

elader at ambion.com elader at ambion.com
Wed Nov 12 22:02:33 EST 1997


In article <regina-0311970922430001 at biomac1.health.ufl.edu>,
  regina at biotech.ufl.edu (Regina Shaw) wrote:
>
> In article <345A61CC.5290 at hotmail.com>, Han <huangz at hotmail.com> wrote:
>
> > Dear netters,
> >
> > I encountered a problem in RNA formaldehyde electrophoresis: I added EB
> > in my formaldehyde agarose gel(final concentration 50ug/100ml) and run
> > gel for 2 hours. Amazingly, when I visualize gel under UV lamp I found
> > very high staining background which evenly distributed and composed
> > almost 2/3 area of gel starting from the margin near the wells. This high
> > flurescent staining is so strong that overhided my RNA bands. I would be
> > very appreciate any suggestion about the possible reasons. My RNA
> > formaldehyde electrophoresis buffers are as following: (please save me
> > out of the nightmare)
> >
> Hi -
> We recently followed New England Biolab's suggestion to do RNA
> electrophoresis in native TBE/agarose gels, with good results. We added
> ethidium bromide both to the gel and to the TBE electrophoresis buffer, to
> a final concentration of 0.5 microgram per ml each. RNA samples were
> heated for 3 min at 65 degrees C in denaturing sample buffer, loaded
> immediately and run for about 1.5 hours. We got good separation of RNA
> bands and very little background. See BioTechniques vol. 9, no. 5 (1990),
> p. 558-560.


Try adding 10ug/ml etbr in your loading dye and not to the gel or the
running buffer. The excess etbr runs out of the top of the gel and there
is zero background staining in the gel. Probably less etbr will also work
ok.

Eric

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