z-suldan at ski.mskcc.nospam.org
Fri Jul 10 12:28:12 EST 1998
In article <wgallin.1250468479A at news.srv.ualberta.ca>,
wgallin at gpu.srv.ualberta.ca (Warren Gallin) wrote:
> In Article <Evvt0x.KFK at liverpool.ac.uk>, fender at liv.ac.uk (fender) wrote:
> >Does anybody out there do alkali transfer for there northerns
> >to nylon membranes and if so what conc of NaoH do you use and
> >what if any post blotting wash?fixing do you carry out?
> >The transcripts I am after are about 5kb and 2kb
> This sounds like a bad idea. RNA is susceptible to base catalyzed
> hydrolysis (the 2' hydroxyl on the ribose gets deprotonated and then attacks
> the phosphate ester next to it), so soaking your gel in NaOH solution is
> probably going to hydrolyze your RNA to very small pieces, possibly too
> small to efficiently hybridize. You will certainly lower your sensitivity
> Warren Gallin
Actually, this is done to hydrolyze very large RNA molecules to smaller
pieces for more efficient transfer.
I've not done this myself as I've gotten efficient transfer up to 8-9 (??)
Kb, o/n, 10xSSC, cappilary transfer.
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