chloramphenicol amplification

mic225p at vaxc.cc.monash.edu.au mic225p at vaxc.cc.monash.edu.au
Thu Sep 24 04:01:29 EST 1992


This is the procedure we use to amplify pUC and pBR based
vectors for large scale plasmid preps. The procedure is
directly from Maniatis.
     1) Innoculate a colony from your plate into 10ml of LB
broth containing an appropriate antibiotic, probably
ampicillin for pUC plasmids. I use universal bottles for this.
Other rich media such as 2xYT, as well as minimal media can
also be used. 
     2) Incubate with shaking at 37 degrees overnight.
     3) Next morning add 0.1ml of your overnight culture to
25ml of LB with antibiotic (ampicillin) in a 100ml flask.
     4) Incubate at 37 degrees with shaking until the culture
is at late log. Usually a couple of hours is enough.
     5) Add 25ml of your fresh late log culture to 500ml of LB
with antibiotic (ampicillin) which has been prewarmed to 37
degrees. Use a 2L flask for good aeration.
     6) Incubate with shaking for exactly 2.5 hours at 37
degrees.
     7) Add chloramphenicol to the culture to a final
concentration of 150ug-170ug/ml. If you use spectinomycin the
final concentration should be 300ug/ml. We have also used
30ug/ml of chloramphenicol and find this is just as good as
the higher concentration.
     8) Incubate with vigorous shaking for 12-16 hours. If
your timing is OK an overnight incubation will do. Don't worry
if the culture turbidity drops during this incubation. But
don't leave the culture for much longer or you may get to much
cell lysis and plasmid yield will drop.
     9) Spin your culture and isolate the plasmid anyway you
like.
     From my reading there is indeed a mutation in pUC
plasmids that increase copy number, but you can still amplify
the plasmids. Most cloning vectors (pUC and pBR included) have
a relaxed replication and don't use the short lifetime host
proteins used in host chromosome replication. So by adding
antibiotics such as chloramphenicol or spectinomycin (which
inhibit protein synthesis) you stop chromosomal replication
and cell division without stopping plasmid replication. The
end result is greatly increased plasmid yeild per cell.

Hope this helps.         Dave  (Monash University)
                         



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