insert-positive blue cols

Jason E Stewart jasons at bmc.uu.se
Thu Jan 21 15:22:55 EST 1993


In article <1993Jan21.090729.79 at ebc.ee>, mspeek at ebc.ee (Mart Speek) wrote:
> 
> 
> Hi Netters;
> 
> I would like to know whether anyone has encountered problems
> in traditional blue/white colony or plaque screening procedures
> (beta galactosidase assay) when sorting out (non)recombinant
> clones. My question is :-( Could relatively long inserts (>0.5kb)
> sometimes give rise blue (beta gal+) clones? How is it possible?
> Any theoretical or practical solutions (incl refs) to this 
> particular problem are welcome.
> 

I have had similar problems with cloning a 242 bp product (i.e. all
positive colonies were faint blue). I pressumed that it had to do with
in-frame insertion, so that the insert left the B-gal in frame while not
disturbing the proteins function. Since I was using a phosphatased vector
cut with two enzymes the background was almost non-existant, so I stopped
using blue-white screening. Perhaps the best recommendation I can make is
to use primers with different enzyme sites and to phosphatase your vector,
that way you won't need screening.

good luck,

jason

----------------------------------------------------------------
Jason Stewart    	                      Father at large
Department of Medical Genetics
jasons at bmc.uu.se



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