[Yeast] odd transformation results

ann.rouse at duke.edu ann.rouse at duke.edu
Wed Mar 22 13:12:41 EST 2006


Hi All,
I've been trying to do a transformation with homologous recombination at a
specific location in the genome.   I'm inserting CaURA3.  I get colonies on
-ura media and get a PCR result the right size with one primer in the insert
and the other next to the insertion site.  The problem is that from the same
colony, I get PCR product for the negative control with the primer next to the
insertion site and a primer for the region that should be replaced.  I working
with a haploid strain (I've double checked by mating the transformants to
tester strains), and I've repeated the PCRs with at least 4 different primers
in each location.  So the seeming result is 2 things in the exact same place in
the genome of a haploid.  Any thoughts or suggestions?
Ann



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