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a strategy for gene relacements

FATIMA at AIMP.UNA.AC.AT FATIMA at AIMP.UNA.AC.AT
Sun Feb 6 11:51:58 EST 1994


Dear Beilin Zhang and other buddies (sorry for the fission yeast community
but I can provide just a S.cer. - specific answer)

- a good general strategy for replacing a wt chromosomal gene by a cloned
mutant allele without introducing markers or rearrangements into the
locus would be:

1. Disrupt the gene of interest with URA3. If you are worried about
sickness or inviability of the disruptant, do it in a diploid or in
the presence of a plasmid-borne wt copy.

2. Transform the URA3 disruptant with a linear DNA fragment carrying
the desired mutation and select for ura- clones using 5-fluoroorotic
acid (FoA). This selection is better to do in two steps, first cotrans-
forming your linear fragment with an excess of a moreless "inert"
plasmid (e.g. YEp13), selecting for the plasmid; in the second step,
replica plate transformants on FoA:

(3. Sporulate and dissect the diploid or get rid of the plasmid 
introduced in step 1.)

Good luck!

Fatima Cvrckova (fatima at aimp.una.ac.at)



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