E.coli transformation - (Feb/25/2008 )

Hello,

I have rescued yeast plasmid using Zymoprep II kit and would like to sequence the gene of interest, how do i do a E.coli transformation of the yeast plasmid? are there any kits available to do so?

Bigsky

Can't you sequence directly from the plasmid you extracted from yeast, using gene-specific primers?
If you really need to transform E. coli (and provided that your plasmid is a shuttle vector), then you need to have chemically competent or electrocompetent E. coli cells (you can buy them or make them yourself). You can transform the chemically competent cells with 1-2 μl of your plasmid. The same thing can be done with electrocompetent cells, but the latter can also be directly transformed by the yeast strain that contains your plasmid by electroporating at two different voltages [i.e. 1.5 kV (25 μF, 100 Ohm) and 2.5 kV (25 μF, 200 Ohm) in a 2 mm cuvette). With the first voltage the plasmid comes out of the yeast cells and with the second it is transformed into E. coli.