site-directed mutagenesis problems

Fiberman wsun at jeeves.ucsd.edu
Wed Dec 9 16:15:26 EST 1992


Hi, netters,

I recently posted the problems I had and appreciate your
responses.  However, I am still not getting the mutants using
the Bio-rad kit.  I am getting transformants with the negative
control DNA - where I make the second strand in the absense of
any mutagenic primer. 

Just recently, I found that my SS DNA was able to transform XL1
blue competents.  Apparently the SS DNA  prep was contaminated
with DS DNA.  Has anyone had experienced this problem?  I am
making SS DNA from the Bluescript plasmid in CJ236 cells.  I am
thinking of gel purifying the SS DNA.  Is this a logical or a
stupid idea?  I appreciate any comments.

-fm



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