site directed mutagenesis problems

Mon Dec 14 18:38:00 EST 1992

I realize this may be a bit late but our mail port was not working 
these messages were returned : D.G.

---------------------fm writes----------------------------------------
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.

I have had similar problems with another strain and got rid of contaminating
DS DNA and RNA by treatment of supernatant with DNase I 10U/ml
and RNase A 10ug/mL for 15 min at 37 degrees C.  Do this before PEG
pptting your phage and your ssDNA prep will be very clean!

Dan Gietz
University of Manitoba

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