large scale B-gal assays?
dalex at NEXUS.MICROIMM.MCGILL.CA
Tue Feb 24 11:51:02 EST 1998
I am screening an E coli plasmid library as part of a search
for a transcriptional regulator.
It's a fairly straight-forward procedure
My gene of interest is hooked up to lacZ in the chromosome
of a delta lac strain and the inserts in the library are cloned
downstream of an IPTG-inducible (tac) promoter.
I am replica plating my colonies on to Xgal media containing
0, 0.1, and 1 mM IPTG. Ideally, expression of the
insert increases with increasing IPTG. If something on the
insert activates my lacZ fusion, then B-gal expression
should increase and my colonies should get more blue.
However, I am not expecting a dramatic increase in expression,
and my X-gal assay does not appear to be sensitive enough.
(i.e. I am going blind attempting to distinguish between blue
more blue, and a little more blue)
I would like to switch to a liquid B-gal assay,
but with 10 000 colonies to screen, the standard Maniatis assay
is not feasible.
Does anyone know of protocol for large scale B-gal assays?
(perhaps using microtitre plates?)
Or has anyone tried new & improved substrates that
provide increased sensitivity on solid media?
Thanks for any advice,
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