dsRNA blots

Juergen Oberstrass oberstra at hrz.uni-kassel.de
Mon Nov 6 05:40:09 EST 2000


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 
configuration.
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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