In article <c601591-2611951026170001 at 184.108.40.206>,
c601591 at mizzou1.missouri.edu (Don Haut) wrote:
> I am having considerable trouble with the Altered Sites II system from
> Promega. My problems are centered around the R408 helper phage. ALL of
> the colonies that I get after using the system are contaminated with the
> R408 helper phage and it is impossible for me to use any of my mutants.
It sounds a bit strange that the R408 is tagging along. Does the same
carry-over occur if you try substituting M13K07 DNA for the R408 DNA? You
could get an idea of K07 carry-over, in this case, by selection on amp/kan
Have you tried transforming ES1301 without addition of the R408 DNA, grow
under ampicillin selection in broth mode, isolate plasmid DNA followed by
transformation into an F- host like DH5alpha and selection on amp plates?
This is pretty much the procedure used for Altered Sites I except that the
in vitro synthesis is done on an ssDNA not denatured dsDNA and I use the
DH5alpha instead of that sickly JM109 strain.
Just some thoughts from a satisfied Altered Sites I user.
tyr-2 at bones.biochem.ualberta.ca