site directed mutagenesis problems

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
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
GIETZ at BLDGHSC.LAN1.UMANITOBA.CA




More information about the Methods mailing list