Q. Double Zap method of yeast-E.coli plasmid transfer??

Graham G.Dellaire at hgu.mrc.ac.uk
Thu Aug 31 03:55:29 EST 2000


Hello All,

I have been trying to do the double zap method of plasmid transfer from
yeast to E. coli (KC8 cells - leu/trp/his, KanR) to rescue library
plasmids.

The conditions I tried where 65 ul of KC8 competent cells with a small
toothpick (~5ul) of a yeast smear mixed before zapping.

First zap was at 1500 volts, 25 uF, 100 ohms
30 sec rest
Second zap 2500 volts, 25 uF, 200 ohms
immediately add 1ml LB and incubate at 37 degrees for 45 minutes
before plating 150 ul of the suspension on M9 -leu plates (plus Amp and
Kan).

I got some background but no "real" colonies, not sure what to expect.

Has anyone else tried this?  What is your experience and conditions for
the double zap if you managed to get the technique to work?

Cheers,

Graham

--
********************
Graham Dellaire, Ph.D.

MRC Human Genetics Unit
Western General Hospital
Crewe Road
Edinburgh, UK
EH4 2XU

Fax: +44 (0)131 343 2620
Phone: +44 (0)131 332 2471
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