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transformation with 2 plasmids

Simon Williams s.p.williams at bioc.cam.ac.uk
Sun Oct 30 23:53:41 EST 1994


>Subject: transformation with 2 plasmids

>Several messages have commented on ways to transform yeast with 2 different
>2-micron plasmids.  I was always under the impression that yeast could maintain
>only 1 2-micron plasmid - that if you transformed with a second one, one of the
>two would be lost or a recombination event would occur to leave only one.


My leu2, ura3 yeast with one LEU2 plasmid and one URA3 plasmid, both 2u in 
origin, are just fine, and I  know other people have them too.  My plasmids 
have only the 2u replication origin though, also pretty common. I transformed 
them with the LEU plasmid first and then the URA, but because I wanted 
the LEU -only yeast too.  No doubt a double transformation would work 
fine.  I (still) use the spheroplasting/CaCl2 treatment transformation.  Maybe 
you run into problems with one plasmid curing the cell of the other if you 
have plasmids based on complete 2u sequence.  Presumably also matters if your 
yeast is cir(o) or cir(+).  So long as you have a selection pressure for both 
plasmids, you should be alright - though the relative copy numbers may not be 
stable.  I think Rose & Broach (Gene Expression Technology pp 234-279, ed 
Goeddel 1990)  is a good starting point for the 2u plasmid stability 
literature.

Simon Williams,
Biochemistry, Cambridge
(s.p.williams at bioc.cam.ac.uk)



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