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pYES with leu/trp marker?

pYES with leu/trp marker? - Yeast Forum

pYES with leu/trp marker? - Discuss research on yeast the model organism. Post questions and topics on the molecular biology and genetics of Saccharomyces cerevisiae and other yeast species.


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Old 09-06-2004, 03:20 PM
David Keszenman-Pereyra
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Default pYES with leu/trp marker?



Quoting Ricky Boernke <ricky_boernke@gmx.net>:



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
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