In article <JCHO.8.0 at bio1.lan.mcgill.ca>, JCHO at bio1.lan.mcgill.ca (John
> yeast biologists,
>> I have a few questions about gene disruption/knockout and was hoping
> someone could shed some light on the matter for me. I've finished making a
> construct for an integrative disruption of my favorite gene(MFG) and am
> transforming presently. The way I made my construct was to cut out the LEU2
> marker (from KS-) and inserted it next to MFG in the ApaI/SalI sites of the
> SK. Cutting this plasmid with SpeI linearizes this SK recombinant vector
> leaving exactly 250 bp of MFG sequence at each end (trust me on the SpeI, it'
> s too complicated to explain). I've done an experiment transforming this
> plasmid DNA into YPH499 which is leu2 and haploid.
Although I may have misread your post, I think you are asking why you got so many non-specific integrations at LEU2 rather than the target locus.
I have played around with using the LEU2 gene for disruption, IMHO the
use of LEU2 (3kb) compared with TRP1 (0.8 kb) increased substantially (order of
100-fold) the number of gene conversions at the marker locus. This could have been the strain I was using (those of the FY series), but I'm inclined to think that LEU2 is not your best bet with less than a kb homology at each end of the construct (I had in the order of 500 bp at each end of my mine and it wasnt specific enough when using LEU2).
Hope this helps;