Library Amplification

Michael Moser 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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