I've been trying to perform a Northern blot for a while now, with no positive results.

I use Digoxigenin labeled PCR products as a probe (after boiling for 5min) in hybridization buffer (50%formamide + 5xSSC, 50mM Na-P, 1%SDS, 10ugssDNA/ml, 5xDenharts) @42*C o/n, dollowed by wash, block in 2%milk 0.3% PBS-T, and incubation with an anti-Digoxin antibody.

I know the transfer works, and that RNA is present after prehybridization (via methylene blue), I also know the antibody works due to dot blotting the DIG-labeled probes.

Does methylene blue staining inhibit probe binding?

Every time Ive tried N.blot Ive stained my membrane with methyl blue before in order to see whether the transfer worked. It's all I can think of at this point, otherwise it has to be my probes.

-CP