protein blocking of DNA/RNA hybridisations
Joao Vasconcelos Costa
jcosta at pen.gulbenkian.pt
Thu Apr 11 07:57:48 EST 1996
Ned Mantei wrote:
> In article <4kisks$gnu at mserv1.dl.ac.uk>, "S. Macfarlane" <mbsmf> wrote:
> > I use Boehringer Mannheim's Blocking Reagent when doing 32-P-labelled
> > Southerns/Northerns. The UK catalogue doesn't say what is in this
> reagent. Does
> > anybody know? Are there any other blocking buffers which don't require the
> > addition of denatured carrier DNA such as Herring Sperm? Can I use fat free
> > milk for this purpose? I seem to remember seeing a description of something
> > called BLOTTO buffer but I don't have any references.
> > Thanks for any help. Stuart MacFarlane, Scottish Crop Research Institute,
> > Dundee, Scotland
> Some things that have been shown to reduce background are
> 1) the components of Denhardt's solution: BSA, Ficoll,
> polyvinylpyrollidone (Denhardt tried everything he had on the shelf (in
> ca. 1966) and these were the things that helped). Non-fat dry milk
> probably replaces BSA with casein.
> 2) 50% formamide hybridization buffer (papers from Bernard Mach somewhere
> between about 1968 and 1975??)
> 3) Lots of SDS (as much as 7%), as in the method of Church and Gilbert.
And also Blotto: 5% nonfat dry milk. Works fine in my lab, although we
don't use it routinely.
***** JOAO VASCONCELOS COSTA, MD, PhD
***** Instituto Gulbenkian de Ciencia - Oeiras, Portugal
***** mailto:jcosta at pen.gulbenkian.pt
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