I am having difficulties transforming an industrial strain of /_S.
cerevisiae_/ by homologous recombination in URA3. I have tried the
Gietz high-efficienty protocol and electroporation (Manivasakam and
Schiestl, 1993). I am selecting for G418 (200ug/ml). None of the
protocols had given me colonies. I am using approximately 5ug of a 5kb
DNA fragment. Is this too much?