moser at U.WASHINGTON.EDU
Wed Oct 12 14:24:52 EST 1994
When we amplify our libraries we always do it on plates to reduce
selection against plasmids which may make E. coli unhappy.
1.) Electroporate using 1 mm gap cuvettes and purchased cells (efficiency
> 1 X 10e9/ mcg) Titer to determine your efficiency. Then do a large
scale transformation using ~ 10 ng per 40 mcl cells.
2.) Plate 1 X 10e6 transformants on 100 mm LB Amp plates. Try to get 10 to
100X the number of original recombinants.
3.) Scrape colonies off plates using LB and disposible cell scrapers ( a
la tissue culture). Suck up liquid using disposible pipets and combine
supernatant in disposible platic contaner.
4.) Isolate DNA using your favorite large scale plasmid prep protocol
(We used CsCl) using only new or disposble implements/tubes.
It is very important to use only equipment that is new/never used when
doing your amplification. If not, you may find yourself cloning lab
plasmids from your library. I cloned S. cerevisiae calmodulin in a
different vector from a S. pombe library the first time I amplified my
library. When I re-amplified and got anal, the background went away. I
just acted as if I was doing PCR. Good luck and happy cloning!
Mike Moser Tel: 206-543-5354
Department of Biochemistry SJ-70 FAX: 206-685-1792
University of Washington moser at u.washington.edu
Seattle, WA 98195 Make peace my beast is yeast
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