IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

a strategy for gene relacements

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)

More information about the Yeast mailing list

Send comments to us at biosci-help [At] net.bio.net