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 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
