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Help, 5-FOA selection of trxs

Steven A. Haney shaney at watson.princeton.edu
Wed Jul 12 19:20:09 EST 1995


In article <199507111439.HAA13763 at net.bio.net>, U09668 at UICVM.CC.UIC.EDU (E
Li) wrote:

> Dear fellow yeasters:
> I have a strain with a genomic disruption of a gene using a URA3 marker, and
> would like to directly transplace the disrupted gene with an intact copy of the
>  gene.  I was thinking about directly selecting the transformants on FOA
> medium, and was wondering if anyone out there has ever tried this.  I don't
> have a selectable marker for the gene I would like to introduce, but I can
> select real transformants as opposed to URA3 revertants, by phenotype.
> Does this approach seem reasonable, and can anyone give me any possible tricks
>  or hints about this strategy? Please e-mail me directly with any advice. Thank
>  you very much.
> 
> 
> E Li
> Univ. of Ill. at Chicago, Dept. of Biological Sciences
> U09668 at uicvm.uic.edu



(This posting is for others that may also be interested.)

URA3 expression cannot be directly selected against because of its fairly
long phenotypic halflife ("phenotypic lag"), and because FOA selection in a
URA3(+) strain is toxic, as opposed to growth static.  Direct selection on
FOA will only identify those cells that were URA3(-) before the
transformation.  

The best solution is to cotransform the gene fragment with a small amount
of a closed circular plasmid with a different selectable marker (eg: TRP1
or LEU2).  Select for the cotransformed plasmid marker.  Then either
replica print to FOA or check the phenotype of the transformants directly,
if you can. I typically get about 0.1% to 1% of the original transformants
that show a gene conversion that removes the URA3 marker.   

Good Luck,

Steven A. Haney
Broach Lab/Dept. of Mol. Biol.
Princeton University
shaney at watson.princeton.edu



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