problems with RNAs denatured with Glyoxal(in Northerns)

upyers mc at u.washington.edu
Tue Feb 27 12:41:22 EST 1996


In article <4gqdq8$gk1 at lyra.csx.cam.ac.uk>, sb at mole.bio.cam.ac.uk (Saverio
Brogna (Genetics)) wrote:

> Please someone,
> 
> could you explaine why when I run Northerns with RNAs denetured in Glyoxal 
> I see two bands from my transcript, and anly one when I used the more 
> commun formaldeyde gels. I dont think that is due to a better resolution 
> of the first method, but I rather think that is an artifact.
> Do you have any suggestions.
> 
> Thanks Saverio
> -- 
> Saverio Brogna
> University of Cambridge, Departement of Genetics
> Downing Street, Cambridge, CB2 3EH, England (UK)
> tel +44-1223-333970, fax +44-1223-333992

I too have seen this. My interpretation is that although glyoxal is a
potent denaturant, it is not as good as fomaldehyde in keeping RNA in a
denatured state. 

You might try heating the samples for a longer time before loading on your gel. 

Also, check the pH of your buffer. If the pH rises above ~8.0 you could
run into problems with secondary structures forming. If this is the case,
provide a circulating buffer system. 

Finally, some manufacturers suggest that you de-ionize your glyoxal first.
I don't know if that will help your problem or not. 

Good luck.

-- 
"The two most common things in the universe are hydrogen and stupidity" Harlan Ellison



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