gene disruption

John Cho JCHO at bio1.lan.mcgill.ca
Mon Mar 4 21:55:18 EST 1996


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. 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.

JOhn



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