Northerns of large RNA?
hurban at cmgm.stanford.edu
Thu Apr 11 10:53:05 EST 1996
In article <4kh90c$2uu at post.its.mcw.edu>, kpleyte at post.its.mcw.edu (Kay A.
> I'm having problems getting a signal for a large RNA (possible 12 kb)
> on a Northern with a 32-P DNA probe. My Northerns work OK for smaller
> RNAs. It looks like the problem may be in the RNA extraction (breakage) or
> the transfer. Any advice for working with large RNAs?
> kpleyte at post.its.mcw.edu
The problem is most likely in the transfer. I use a fairly standard
Formaldehyde gel protocol (MOPS buffer). To aid in the transfer of large
RNAs, after running the gel I soak it in 50 mM NaOH, 100 mM NaCl for 45
min, followed by soaking in 100 mM Tris pH 7.5 for 30 min. I then
transfer O/N in 20X SSC as usual. This alkali treatment does not seem to
affect subsequent hybridization of probe but does apparently break the
larger RNAs into smaller chunks that transfer more readily. I hope this
helps you out and good luck with your work.
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