Hi out there
I have run into a problem which I would appeciate some help with. I am using
a PCR gene disruption method (oligos which amplify the his3 gene to give a product with 50 bp homology to the target gene at each end). This method requires the use of a his3 deletion strain (to reduce integration at the his3 locus). I am using his3D200 allele, but found recently that this is associated with a petite phenotype (since the his3 deletion removes some of the promotor of the adjacent pet56 gene) - so if I use a diploid his3D200 strain I cant sporulate it! Has anyone else come across this problem?
Anyway, I am going to try the disruption in the diploid, then try putting the pet56 gene in on a plasmid. Does anyone have a clone of the pet56 gene? Would much appeciate the gift if it is available.
Thanks in advance,
macdiarm at sbsu1.auckland.ac.nz