Getting glyoxal out of RNA

luc luc at sgi.celgen.kuleuven.ac.be
Wed Nov 4 11:49:37 EST 1998


after blotting to a positively charged membrane, put the membrane for 10 min
on 0.1M NaOH, then crosslink at about halve the normal intensity.
luc

Kathy Wood wrote:

> I am trying to find the best way to normalize transgenic tobacco RNA
> samples by equalizing rRNA levels.  Our lab prefers to use glyoxalated
> RNA run on agarose gels rather than formaldehyde denatured RNA.  One
> problem I have noticed is that if I run unglyoxalated RNA (that has been
> heat denatured and rapidly cooled) on an agarose gel alongside
> glyoxalated RNA, the eventual probe signal for the glyoxalated RNA is
> significantly reduced as compared with the unglyoxalted RNA on my
> northern blot.  Obviously the glyoxal is interfering with probe
> hybridization even though I have tried to remove the glyoxal by
> incubating the UV-crosslinked blot with 20mM Tris-Cl, pH 8 at 65oC for
> 15 minutes.  After this incubation, I continue with the hybridization
> protocol.
>
> Does anyone have a better method of removing glyoxal or otherwise
> optimizing this signal?




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