Saccharomyces transformation

Tom Wehrer seq14 at sfsuvax1.sfsu.edu
Thu Jan 26 02:19:28 EST 1995


I have been attempting to transform two separate haploid strains of S.
cerevisaie with a 5.5 kb linearized plasmid. The strains are ura- and my
plasmid contains a ura marker. I have been using the spheroplast
transformation method and plating transformants on -ura SC/1 M sorbitol
plates. My plasmid also contains sequences flanking and containing
portions of the 5' and 3' ends of the RPD3 gene (required for mitotic and
meiotic recombination). I am hoping for an integration of my plasmid into
the wt RPD3 gene, disabling it. However, I have been having problems with
the transformation - I have gotten growth on my negative control plates
several times (transformed with sonicated salmon sperm DNA only). If
anyone has any suggestions as to what might be happening, or ways to
improve sticky portions of the protocol, please reply of mail me at:
            emery at sfsuvax1.sfsu.edu

   Thanks-
               -Emery Dora



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