Dear Yeast People,
I use a PCR URA3 linear cassette to disrupt an essential gene in diploid
yeast. This yeast strain has a point mutation in URA3 gene and turns out
a URA- background. The disruption of this gene is lethal in haploid yeast
cells.
Theoretically, I should at least get some transformants which remain only
one wild type allele. I have been working on both Lithium Acetate and
electroporation transformation methods for a while, but these methods never
bring me the right transformants. I always get few transformants with the
homologous wild type allele but never get the heterozygous one with one
wild type and one marker exchanged allele.
So a question comes out of my mind. Is it possible that the phenotype is
lethal even with a wild type allele in diploid yeast cells? Could any one
suggest me some relevant references in this case?
So many thanks!
Yiling
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Yiling Chen
Department of Botany, Box 355325
University of Washington
Seattle, WA 98195-1800
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