Dear yeast community,
I have been using 5'FOA selection for ura- strain in pombe by
transforming cells with a linear fragment of an epitope tagged gene X
flanked by its genomic 5' and 3' UTR sequences. I'm hoping to replace
the ura4+ gene that has been placed in the gene X locus previously with
this epitope tagged gene X through double crossover of homologous
recombination, thus yielding the ura- progeny. So far I have no success
whatsoever, i.e. no transformants at all when selected on 0.1% 5'FOA
plates. I was told that cell viability on FOA plates is low, question
is how do I circumvent this problem. Does anyone know what is the
frequency of obtaining a positive clone through such a transformation?
Any suggestions? Please help.
Thanks,
Wenyi Feng
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