Can anybody reccommend a protocol for knocking out Ura3 with FOA
I'm trying the 2nd step of a 2 step gene replacemnt in a YAC where
Ura3 was the 1st. I've already completely deleted Ura3 from the arm, and
I reintroduced it within the YAC insert I got high efficiency (10^5,
LiOAc) and bona
fide replacement, but when I try to replace Ura3 with MFG, I get 10^2 and
stimulation over background reversion (loss of Ura3). The homologous
the same in both steps.
Qs: Is 10^-5 high for background Ura3 loss?
Do I need to do anything special to the cells before plating to allow the
Ura3 KO to occur, before the FOA hits them?