High background in Southern hybridization

[Ned Mantei]

In article <8jesc9$nt3$1 at oravannahka.helsinki.fi>, Mikko Taipale 
<mikko.taipale at helsinki.fi> wrote:

>Do you also use charged nylon and alkaline blotting? With positively
>charged membranes, I believe there is no need for additional crosslinking
>provided that the blotting is done with an alkaline solution.

We get very good results Hybond N+ with alkaline blotting. As mentioned 
by Mikko, cross-linking occurs during the alkaline blotting process. Two 
not-so-intuitive things have been found to lower background in our hands:
1) Don't let the membrane dry between blotting and pre-hybridization. I 
rinse 2x in 2x SSPE after blotting and then put the blot in a bag and 
start the prehybridization immediately.
2) The capacity of the membrane seems to be extremely high, and you need 
to use enough prehybridization and hybridization buffer to saturate the 
non-specific binding capacity (ca. 0.1 ml per square centimeter). 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 think hybridizing in plastic bags is superior to hybridization 
tubes. Shaking or other movement during hybridization is unnecessary. My 
final wash is with 0.5X SSPE--0.2% SDS in a plastic bag submerged in a 
65 C water bath.)

Something that has also helped a lot (but not because of background) is 
to use Kodak BioMax MS film and screen. The sensitivity is so high with 
this combination that the bands are overexposed after 48 hours.

-- 
Ned Mantei
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland