Selection for large inserts in plasmids?

Dom Spinella dspinella at chugaibio.com
Fri Mar 13 10:25:02 EST 1998


Dear BioNetters:
My lab is developing a technology in which it would be of value to
shotgun clone sequences of varying lengths into a standard plasmid
vector, but have some sort of selection for insert sizes greater than
about 500 bp -- without having to perform screens on individual
colonies, and without size-selecting the inserts prior to cloning.  Does
anyone know of a method for doing this?  One approach I thought about
was to engineer a cloning site between the B-gal promoter and the full
length B-gal structural gene (derived from a plasmid such as PCH110). 
The thought here is that separating the promoter elements from the
structural gene by more than a certain minimal distance might abrogate
expression of B-gal and lead to white colonies on X-gal plates. 
However, I don't know if this will work, or how far apart the promoter
and structural gene have to be to reduce transcription levels.  Anyone
have any thoughts? Any ideas would be welcome. Thanks!
--Dom Spinella



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