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kanMX4 deletion mutants

Trey Ideker trunk at u.washington.edu
Mon Jan 10 17:11:04 EST 2000


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
the transformation:

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.

Thanks, Trey

Trey Ideker
UW Molecular Biotechnology
206.616.5117 [WORK]   206.284.2496 [HOME]
trunk at u.washington.edu


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