Dear Netters,
I'm having trouble with yeast Knockout (KO). I have PCR'd a fragment of
Kanamycin Resistance gene with two 30 bp's at both ends which is identical
to the both ends of my target gene
And I followed well-established LiAc transformation method to introduce
this 1.6 kb DNA fragment. Since transformation efficiency and
recombination probability would be very low for this case, I made ten
transformation plates for each haploid and diploid strain.
According to the comment from my colleague who has done this kind of
experiments, I incubated yeast cells in 5mL YPD (without G418 selection)
for 5 hours in a roller drum, and plated on YPD + G418 selection media at
the end of experiment.
5-hour pre-incubation seemed to allow recombination and expression of
kanamycin resistance gene. Here is my suspect. Since the doubling time of
my yeast strain is about 2 hours, is it necessary to incubate for 5 hours.
I don't know how long it takes to express Kan-R gene, but I'm worried that
for too long incubation, untransformed cells might outgrow transformed
cells.
Thanks again in advance for any comments which would be welcomed to
nc1 at acpub.duke.edu
Namjin Chung
Program in Molecular Cancer Biology
Department of Cell Biology
Duke University Medical Center
Durham, NC 27710
