Question on Liquid vs. Solid Media with Invitrogen's TOPO-10 bacteria!

Dr. Duncan Clark duncan at nospam.demon.co.uk
Thu Jul 9 04:23:22 EST 1998


In article <1998070901042300.VAA27304 at ladder01.news.aol.com>, Gregg Silk
<greggsilk at aol.com> writes
>Also, the higher amounts of yeast extract in LB is bad for
>cells.

So explain why cells grow to higher cell densities using terrific broth
which contains 24g/litre of yeast extract as opposed to 5g/l in LB. OK
there is glycerol, phosphate buffer in TB but from your statement this
level of yeast extract would be even more detrimental. In reality the
opposite is true. I'm not saying that TB is the best growth media for
E.coli but your statement that yeast extract is bad is misleading.

As to the original question. E.coli will tolerate from 18C to 43C and pH
6.5 to 8.0 without problems. I would suggest that you are actually
trying to grow on non-recombinant ie Amp sensitive colony in Amp
containing media. I would make up your plates fresh remembering to add
the ampicillin to 100ug/ml at a temperature of the media no higher than
45-50C. Any higher and you will kill the Amp. Also use fresh stocks of
Ampicillin. Even better is 80ug/ml methicillin and 20ug/ml ampicillin
but you could have enormous problems trying to locate methicillin as it
has been withdrawn from general sale. 

Duncan 
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk



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