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. I've obtained
approximately 6000 colonies which grow well on SC-leu selective media. What
concerns me are the control plates. Untransformed YPH499 yielded no colonies
as expected. I am troubled by what happened when I also transformed Sal I
linearized KS LEU2 (without the MFG sequence) - these plates show roughly
the same number of viable colonies as my other plates with MFG LEU. I
understand that a double recombination event may account for some of these
transformants, but given that the linearization of this SK LEU plasmid is
not a digestion at a site internal to the LEU2 gene, would any of you say
that the number of transformants obtained is highly unusual? Have I made
some sort of fundamental error in my understanding of yeast recombination?
Given that I am using Bluescript as the plasmid, I don't believe that my
vector contains an ARS sequence, nor do I believe there are any Ty elements
upstream of the LEU2. Was this an experiment gone bad? I'd just like to know
if I've made any reasoning errors before I start screening for the
disruption. Any thoughts would be much appreciated.