Altered Sites II

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Sun Nov 26 17:59:19 EST 1995


In article <c601591-2611951026170001 at 128.206.12.143>,
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. 

Don,

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
plates.

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.

Cheers

Karl

-- 
Karl Fischer
tyr-2 at bones.biochem.ualberta.ca





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