Help Northern Blocking!

[Gene Huh]

In article <4prqbq$473 at hobbes.cc.uga.edu>, watson at dogwood.botany.uga.edu
(Mark Watson) wrote:

> Hello Net People,
> I was wondering if any of you folks have had or seen this problem.  It has 
> arisen suddenly and I can't seem to figure out what is going on.  I have had 
> horrible non-specific background on my last couple of northerns.  It
seems the 
> probe is binding to the filter and not the RNA.  I remove the unicorporated 
> nucleotides from my random primer labelled DNA probe.  I blocked overnight 
> with 5X SSC + 5X Denhardts + 1% SDS and 100 ug/ml sheered/denatured Herring 
> Sperm DNA + 50% formamide (biorad Mol. Biol. Grade) at 42oC.  I have been 
> using one of those rotary hyb ovens.  The nonspecific background can be
as bad 
> as to show the RNA as a negative image.  Please help before I go crazy!
> Thanks
> Mark
> 
> ps  I have been blotting to biorad zeta probe nylon with 10X ssc
> 
> 
> *******************************
>  Mark Watson
>  watson at dogwood.botany.uga.edu
> *******************************

The latest Zetaprobe manual suggests something like 50% formamide, 7% SDS,
0.25M NaPO4 (0.25 wrt Na+) for both prehyb and hyb, with a 5%
SDS-containing first wash.  This is pretty close to Church/Gilbert
hybridization conditions and, for me,  seems to work well with a variety
of membranes; if you call biorad you could probably get them to fax you
the manual (at the cost of getting on their mailing list, I suppose :)  I
usually throw in 5X Denhardt's (0.1% each of BSA, PVP and Ficoll) in the
prehyb/hyb/first wash, since I see this reducing background, too.

Dr. Gene Huh
UCBerkeley, Dept of Molecular and Cell Biology
gshuh at garnet.berkeley.edu