Small RNA electrophoresis: any tips?
nicholas_theodorakis at urmc.rochester.edu
Tue Oct 5 08:55:23 EST 1999
In article <JM-0510991506290001 at macbb2212b.unil.ch>,
JM at 843724082793.unil.ch (Jerry M.) wrote:
> I've looked in the archives before posting this question, but I
> find a satisfactory answer: what's the best way to do a nice RNA
> electrophoresis to just show the size of a small (200 bp)
> transcript? I'm
> not into doing Northerns, I just want to take a nice picture of a
> gel. Since the gel must be denaturing, I've tried MOPS/Acetate
> buffer +
> 2.2 M formaldehyde + EtBr and the background fluorescence is a
> disaster no
> matter what the EtBr concentration is, however I could faintly see
> bands of the marker. I've tried adding EtBr in the sample and not
> in the
> gel: the small RNA doesn't seem to stain well enough to see
> unless there's a huge amount (>5 ug) of it, and then there's
> which couldn't really be called a band. I've tried to lower the
> formaldehyde to 0.22 M: the contrast is much better but apparently
> not denaturing enough (several upper bands appear instead of just
> one). I
> don't know what else I could try. Methylene blue staining? Or the
> dye (sorry, can't remember the name righ now)? Would
> gels be better? Any tip would be much appreciated.
> Thanks in advance,
> Jerry M.
> Please do not reply by e-mail: address not valid.
Polyacrylamide in urea/TBE is what you need. Use 4-6% acrylamide, but
the 4% is probably too hard to handle. IIRC, xylene cyanol runs at
about 150 or so nt. in 6% acryalmide, so make sure to run the gel long
enough. You can stain with EtBr after electrophoresis (0.5 - 1 ug/ml in
TBE) for about 15 min. You don't need to destain (never tried other
fluorescent dyes, but I don't see why they won't work). The background
will be very low, and the bands should be sharp - you should get a
The gel might be a little tricky to handle. I keep one of the gel
plates underneath it. To photograph, lift the whole plate and gel up,
cover with Saran wrap, and flip it over onto the transilluminator. You
can take a picture right through the wrap.
If you need to probe it, you can electroblot it onto charged nylon, but
use 1/2 X TBE instead of 1X, because the current draw will be high.
(You might want to blot in the cold room to keep it from heating too
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