Clontech's Transformer Kit- Help please

Dave Boyd dboyd at ccu.umanitoba.ca
Thu Sep 1 11:02:00 EST 1994


We want to use Clontech's Transformer site-directed mutagenesis kit but
have been having some difficulties with it. After having used it once on
our own construct unsuccesfully, we decided to go through the control
reactions in the kit; this is not working well at all. Basically at the the
end of it all  we get very low numbers of colonies and only about 30%
mutants of those we do get. When we plate out 100 ul of the transformation
mixture we are getting only 0- 20 colonies on a plate; this sucks, I am
expecting at lot more than this. We use electroporation for transformation
and are cells are fine; lots of colonies with uncut plasmid.

Questions; 1) How efficient is the mixed polymerase/ligase reaction? It
seems to me if this step isn't working well then your fucked from the
beginning.
                  2) Is it possible during the first restriction (to reduce
the number of parental plasmids) that the enzyme could cut the heteroduplex
at its original site thus reducing the number of mutant plasmids? Can you
skip this initial restriction? 
                   3) Is the mutS mutation in the E. coli strain they give
you in the kit stable even in the presence of tetracyline?

Thanks for any help we receive!

p.s. I did read David Adelson's post about his successful use of the kit
first time. Lucky sod. Must be the all the sunshine downunder.

p.s.s What the fuck anyway! We pay the outrageous price for these kits
because of the convenience. Fine, but Iexpect the fucking thing to work at
least somewhere in the ball park the way the company claims it does. 
To err is human; to forgive, unlikely.



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