pYES with leu/trp marker?

David Keszenman-Pereyra Keszenman-Pereyra at sheffield.ac.uk
Mon Sep 6 06:18:09 EST 2004


Quoting Ricky Boernke <ricky_boernke at gmx.net>:

> Dear yeasters,
>=20
> I'd like to epxress two proteins at a time in yeast. I'v got one in
> pYES-DEST from Invitrogen which carries a ura marker. I'd like to know
> whether any would have a version of pYES or any other expression
> plasmid with GAL-promotor carrying a leu or trp marker?
> Thanks
> Ricky


Ricky,

Swap the marker (see YEAST 13: 647=96653, 1997; 20: 985=96993 2003).

Here is an "simple" procedure:

Design primers with sequence identity of the URA3-pYES-DEST region
(for example 60 mer) and a 27-30 mer extension of the new marker (for
example LEU2).

PCR using as a template a plasmid carrying LEU2 without homologies
with pYES-DEST.

Co-transform a yeast strains deleted in URA3 and LEU2 with PYES-DEST
and the PCR product. Select LEU+ colonies (another option: first
transform the strain with PYES-DEST and then transform the
strain+pYES-DEST with the PCR product.

Retrieve plasmid in E coli.


David=20

   =20
David Keszenman-Pereyra
University of Sheffield
Department of Molecular Biology and Biotechnology
E28, Firth Court, Western Bank, Sheffield S10 2TN
UK
Telephone:(44)01142224193
Fax:      (44)01142222800
Email: Keszenman-Pereyra at sheffield.ac.uk


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