Help with screening for DNA-binding proteins

Leonard N. Bloksberg bloksber at pilot.msu.edu
Tue Nov 2 13:33:00 EST 1993


In Article <simmenlab.11.752248027 at animal.ufl.edu> "simmenlab at animal.ufl.edu (Dr. Simmen's Lab)" says:
> Hello netters,
> 
> I apologize up front if this has already been discussed in this group, but 
> we are new at this (screening and internetting). The problem we have is that 
> we want to clone a DNA-binding protein from either a lambda gt11 (random 
> primed) or lambda zap library (oligo dT primed). As a 
> probe we are using concatenated oligonucloetide that encodes for a protein 
> binding site. To screen we follow the Vinson et al. protocol with the 
> modifications from Singh et al. That includes a denaturation with 
> guanidine HCl and stepwise renaturation of the filters. 
> 
> The first time we tried this method, we pulled out several positives but now 
> we can't seem to get positives any more. We know the oligo binds essentially 
> one polypeptide (from southwesterns and uv x-linking) and the libraries are 
> good.  Does any body have any suggestions. Is there any trick to the 
> denaturation-renaturation steps? We perform all these steps at room temp. Do 
> people use higher salt concentrations? (The buffer calls for 25 mM NaCl)
> 
> Any help will be appreciated. If you want you can write directly to us and 
> if there is an interest, I'll post it later.
> 
> Thanks, Beatriz.
> 
.
Hi Beatriz We, too are interested in any info you may get.  Please post a 
summary either to the net, or to my email address.
.
.		Leonard N. Bloksberg
.		Bloksber at pilot.msu.edu
.
.



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