A good method for nested deletion mutants?

S. J. Mc Mahon sm010b at uhura.cc.rochester.edu
Mon Jul 24 14:20:45 EST 1995


Howdy everyone,
	I was just wondering if anyone knows a good method for generating
nested deletion mutants.  I am working with the Puc18 plasmid, which
contains a 457bp insert that I wish to mutate.  The first 301nt of the
insert contain the coding region and the last 157 are 3' untranslated.  If
possible I would like to remove the 157 3' untranslated region completely,
and remove about two amino acids worth at a time from the 3' end of my gene,
for a total of about 15 to 20 mutants each shorter than the other by about 2
aa. I also need to cut/remove the insert from the plasmid, and clone it into a
GST expression vector, PGEX-2T, but due to some modifications made to this
plasmid previously to aid in purification and tagging of the protein, the
only restriction site available for sub-cloning is Eco RI which is found
internally in my insert.  I would like to avoid partial digestions if
possible so if anyone has any ideas about this let me know.  Much
appreciation.


Sean J. McMahon
University of Rochester
sm010b at uhura.cc.rochester.edu



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