Doug Stemke dstemke at umabnet.ab.umd.edu
Mon Jul 8 12:01:02 EST 1996

On Tue, 2 Jul 1996, Nitin S. Baliga wrote:

> We have been facing a strange problem in cloning DNA fragments off-late.
> Analysis of recombinants (after blue-white screening) reveals presence of
> a range of deletion forms of vector DNA. We are wondering if this problem
> is unique to the vector DNA we have been using. If anyone else has faced
> this problem please let me know. (The vector which gave us the problem is
> a shuttle vector for Halobacteria and E.coli; a derivative of pKSII).
I did run into similar problems when cloning into B. subtilis with very 
strong promoters.  It seems that the recombination system in this 
organism very efficiently removes sequences it can't handle.  It is also 
possible, although unlikely, you have a methylation incompatibility 
problem (I say unlikely as I suspect the entire vector would have been 
chewed up).  You might try cloning into a lower copy number vector, that 
sometimes helps.

Good luck.

Douglas Stemke

Biological Process Technology Institute
University of Minnesota

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