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FOA blues.....

Anthony Tomlinson tomlinson at pplros.demon.co.uk
Mon Jul 21 09:14:19 EST 1997


Can anybody reccommend a protocol for knocking out Ura3 with FOA
selection?
I'm trying the 2nd step of a 2 step gene replacemnt in a YAC where
insertion of
Ura3 was the 1st.  I've already completely deleted Ura3 from the arm, and
when
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
no
stimulation over background reversion (loss of Ura3).  The homologous
regions are
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?
	

Thanks,

Anthony



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