High background in Southern hybridization

R. John Lye rjl6n at virginia.edu
Thu Jun 29 06:24:01 EST 2000


Ned Mantei wrote:

> It's
> necessary to use fresh buffer for the hybridization itself; don't just
> add probe to the prehyb buffer. (I use buffer with 6X SSPE, 50%
> formamide, 5x Denhardt's, 0.5% SDS, and 250 ug/ml denatured salmon sperm
> DNA).

I've gotten much better results by switching to the "Modified Church
and Gilbert Buffer" that Amersham recommends for Hybond
(0.5 M NaPO4 pH 7.2, 7% SDS (w/v), 10 mM EDTA) - and it is much
simpler to make up, as well.

John






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