I am making point mutation in yeast cells using two-step transplacement method: integration of a Ura+ plasmid containing the gene mutation and then pop-out one copy by FOA selection. The integration step seems fine because there are many Ura+ colonies and allele specific PCR verify mutation is integrated. However, after culture the Ura+ colonies O/N in YEPD and then plating on FOA plates only wild-type allele is there. It looks like the mutant copy of gene is always popped out.
The integration plasmid works perfectly before. The problem only showed recently. So what could be the problem? Thank you!
Is the mutation in the center of the region of homology or close to one end? This may be merely the result of size and homologous regions.
That may be the problem although then you wouldn't be getting the wild type back but rather the integrated plasmid. Again if the mutation is located very assymetrically in the gene that could also explain skewed results.
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