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
Dear Emery,
A few questions come to mind:
1. Is RPD3 essential for growth? If it is, then you should not expect to
recover a disruption of the gene in a haploid.
2. Do you get URA+ transformants with another carrier (eg. tRNA)? If so,
your ura3 allele may be reverting at a high frequency and may not be
suitable as a selectable marker.
David Jansma
jansma at sickkids.on.ca
