As a quickie solution - you could just get those deletion strains from
either ATCC or Research genetics. (follow the link off of the SGD site)
I just checked and they have the GAL3 deletion
3949 YDR009W GAL3 BY4741
But I don't know if the GAL1 deletion is available yet.
On the actual deletion themselves - I can't offer much direct help. I
would try increasing the size of the flanking sites of homology. (order
longer primers). I don't know why they are non-maters now - did you go
back and characterize their genotype?
In article <85drq8$8a$1 at mercury.hgmp.mrc.ac.uk>,
Trey Ideker <trunk at u.washington.edu> wrote:
>> I am attempting to delete several genes involved in galactose
> 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
> 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
>> Confirmation PCR indicates that both wt and kanMX-transformed copies
> 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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