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

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Thu Sep 7 19:21:17 EST 2000


In article <39AE1D7B.154ED13D at hgu.mrc.ac.uk>, G.Dellaire at hgu.mrc.ac.uk (Graham) wrote:
>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? 

Yes, I vaguely recall a number of bacteria-to-bacteria and yeasts-to-E.coli
applications published.

> What is your experience and conditions for
>the double zap if you managed to get the technique to work?

One thing to remember is that "double zap" is very inefficient, so you might
just have a problem with not having enough transformants. I don't know, 
off hand I would try mixing well-washed E.coli and yeast "electrically
competent" cultures 1:1 (5 ml culture is more than enough for a single 
such prep; washing can be done within 30 min) and giving only one
pulse but of high intensity. Perhaps 1.8 kV in 0.1 cm cuvette (18 kV/cm). 
Yeasts are a lot bigger than E.coli and this field should blow them apart. 
Some of the plasmid should be able to reach E.coli withing the duration 
of the pulse (electrotransformation is basically a forced electrophoresis
into small holes created in memebranes by dielectric breakdown). 

        - Dima






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