cDNA expression cloning in pET vectors

William F. Wade William.F.Wade at dartmouth.edu
Wed Dec 20 15:47:09 EST 1995



We are trying to clone a protein that is required for  class II signal
transduction on murine B cells.

Using a cDNA library from a B cell tumor that constitutively expresses
the protein, we thought we would clone the cDNAs into pET 11a-c.  The
control transformations work according to Stratagene specifications (1
x 10^6 colonies/ug of DNA), but I was wondering if anyone knows of a
way to increase this efficiency that stratagene may not know about. 
Using the Bam digested pET vector that is kinased and ligated back to
itself we get around 5 x 10^5 colonies/ug of DNA; so it seems the
vector is ok.  The background for the Bam digested vector is 1-2
colonies/150 ng of transformed DNA.

We are having a really difficult time ligating our cDNA into the pET
vectors.  Does anyone know of this system and any tricks that may be
required to push this ligation to work?

Can those of you that read this board tell me how many colonies to
expect with real world cDNA cloned into bacterial expression vectors? 
I am trying to understand what is the best that can be expected so we
don't waste time optimizing and can get on with doing a bunch of
transformations.  We have devised a primer to amplify the cDNA after
the blunt-end Bam linkers have been added.  This was designed to
increase our linker tailed cDNA in case that was a limiting step.

Any advice or '"rules of the road" for cDNA expression cloning using
the pET vectors would be appreaciated.  Either post answers to this
board of my email address, william.wade at dartmouth.edu.

Thanks for your help,

Bill Wade, Ph.D.
Assistant Professor
Dartmouth Medical School



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