DNA Probe for [32]P northern blotting

ranen aviner via methods%40net.bio.net (by ranen.aviner from gmail.com)
Sun Jul 18 05:42:40 EST 2010


Hello,
I recently encountered a problem working with [32]P labelled DNA probes for
RNA detection.

I pre-hybridize positively charged nylon membrane in 7% SDS 0.3M Na2PO4 for
several hours, then add my labelled probe (purified using a PROBER column)
for overnight incubation, followed by washing in 5% SDS 20mM Na2PO4.

This used to work fine for my purposes, but now I repeatedly get very hot
membranes and the radioactivity simply won't go away, even after extensive
washing (and stripping under harsh conditions). I assume this may be due to
non-specific binding of labelled nucleotides, but I fail to understand why.
Any ideas?

Thanks!
R. Aviner


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