insert-positive blue cols

Jason E Stewart jasons at
Thu Jan 21 15:22:55 EST 1993

In article <1993Jan21.090729.79 at>, mspeek at (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 Stewart    	                      Father at large
Department of Medical Genetics
jasons at

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