I am attempting to delete several genes involved in galactose induction,
namely GAL1 and GAL3, in the MATa haploid strain BY4741. For some
reason, although the strain I am starting with is haploid, all the
transformants I obtain appear to be diploids. Can anyone help me figure
out what is going on? (I am at my wit's end here.) Some facts about
I am using a kanMX4 cassette to perform the disruption, amplified with
40bp of homology to either the GAL1 or GAL3 genes on either end.
I am using the standard Geitz transformation protocol, followed by
outgrowth on YPD and then plating on YPD+G418.
I have tested that the cells I am transforming are haploid, MATa.
After transformation and selection on G418, I obtain 10-20 healthy
looking colonies per plate, but all of these are non-maters when tested.
Confirmation PCR indicates that both wt and kanMX-transformed copies of
the loci are present in my transformants.
Sequencing of these confirmation PCR bands indicates that I am indeed
amplifying the correct locus.
Any advice that anybody can send would be greatly appreciated.
UW Molecular Biotechnology
206.616.5117 [WORK] 206.284.2496 [HOME]
trunk at u.washington.edu
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