High background in Southern hybridization

Mikko Taipale mikko.taipale at helsinki.fi
Wed Jun 28 03:54:01 EST 2000


I've had annoying problems with high background in standard Southern
hybridization. I have digested 5 ug of human genomic DNA w/ EcoRI, BamHI
and HindIII and used several different probes (500 bp, no Alus or other
repeats, labeling with Amersham RediPrime II), but the signal is always
very weak compared to the background smear. Hybridization is at 60-72C,
first wash with 2xSSC, 0.05% SDS at 65C.

I've used alkaline blotting (0.5M NaOH, 1,5M NaCl), hyb solution is simply
10% dextran sulphate, 1M NaCl, 1% SDS (works well with Northerns
etc.) with 0,15 mg/ml herring sperm DNA. Addition of Cot-1 DNA (1
ug/ml) does not seem to reduce background, commercial ExpressHyb solution
does not give any better results, either. Has anybody had similar
problems? Do hybridization solutions with formamide give usually any
better results?

Mikko Taipale
Univ. of Helsinki






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