wgallin at gpu.srv.ualberta.ca
Mon Nov 6 12:11:14 EST 2000
RNA is subject to base catalyzed hydrolysis, courtesy of its vicinial
diols. I would guess that you are hydrolyzing most of your target
during your denaturation and immobilization steps.
Juergen Oberstrass wrote:
> In article <mike.rott-0511001446020001 at ppp285.urz.uni-heidelberg.de>,
> mike.rott at tuebingen.mpg.de (Mike Rott) wrote:
> > I am having problems with dsRNA blots. I use a nondenaturing TAE agarose
> > gel. treat in 50mM NaOH for 30 min and then blot with 20xSSC to charged
> > nylon membrane. Membrane is then treated for 10 min in 50mM NaOH to fix
> > and denature. Using this method with either dsDNA or ssRNA results in
> > excellent blots but not from dsRNA. I assume that 50mM NaOH is not
> > sufficient to denature dsRNA and am going to try a higher concentration.
> > In the meantime what have others tried that worked.
> > mike
> I had a similar problem. dsRNA is the most stable nucleic acid
> One alternative is to change the gel system to phosphate buffered
> polyacrylamid gels which unfortunately need buffer circularization. But
> then you can denature the gel in glyoxal prior blotting and transfer the
> denatured RNA.
> Alternatively you can run a denaturing gel. If you need the information
> if the RNA was double stranded you hybridize subsequently with strand
> specific probes.
> Good luck, Juergen
> Juergen Oberstrass E-Mail: oberstra at hrz.uni-kassel.de
> Kassel University Genetics Department
> Heinrich Plett Str. 40
> D-34132 Kassel / Germany
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