pYES with leu/trp marker?
Keszenman-Pereyra at sheffield.ac.uk
Mon Sep 6 06:18:09 EST 2004
Quoting Ricky Boernke <ricky_boernke at gmx.net>:
> Dear yeasters,
> 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?
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
PCR using as a template a plasmid carrying LEU2 without homologies
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.
University of Sheffield
Department of Molecular Biology and Biotechnology
E28, Firth Court, Western Bank, Sheffield S10 2TN
Email: Keszenman-Pereyra at sheffield.ac.uk
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