Mutagenesis Blues

Yasha Hartberg Yasha at
Sun Oct 29 14:26:19 EST 1995

Our group is trying to use Kunkel mutagenesis to create a number of
site-directed mutants, a method which has been working in our hands for a
number of years.  The past year, however, we have not had any success. 
Despite a number of efforts, we are never able to get any plaques after
extension and transformation.

Sequencing from our template has told us that the template is good and
that the primers hybridize to the correct sequence.  End-labeling the
primer with P-32 has told us that our kination is working. 
Direct-incorporation of P-32 in the extension reaction has told us that we
are getting extension.  Controls have shown us both that the bacterial
strain (a JM103 derivative) is competent and that it contains the
F'-factor.  In short, each of the individual steps seems to be working
fine so why in the hell can't we get any plaques?!!!

Any suggestions (yes we could change techniques but this has worked so
well for so long) would be greatly appreciated.

Yasha Hartberg
Texas A&M University
"Now ritual is the husk of faith and loyalty, the beginning of confusion."  
Lao Tsu

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