pET and ampicillin
jhartley at access4.digex.net
Tue Nov 15 06:44:06 EST 1994
We have used methicillin for several years to inhibit lactamase.
We use 100 ug/ml amp and 200 ug/ml methicillin in plates, but I
understand that some use 20 amp and 80 meth. Methicillin is much cheaper
than carbenicillin, and does a good job of suppressing satellite
colonies. I'd be careful using it in broth cultures-I tried it once and
I thought my miniprep yields were low.
Life Technologies, Inc.
Gaithersburg, MD USA
On 10 Nov 1994, Todd Miller - Pharmacology wrote:
> Our lab is using the pET system to express eukaryotic pro-
> teins. We seem to be experiencing a problem with the BL21 host cells
> losing their plasmid. Does anyone have suggestions on how to avoid
> this? We're thinking of trying carbenicillin but its about 10 times
> as expensive as ampicillin. Also, have inhibitors of beta lactamase
> ever been used (like clavulanic acid)?
> I'm aware that overnight cultures should be highly diluted and
> we've also played around a bit with the pLys S and E containing hosts.
> We're trying to use this system in a fermentor, but I fear that if we
> grow to the high densities that can be achieved in a fermentor, none
> of the bacteria will have the plasmid when we induce expression.
> Thanks for any suggestions.
> Todd Miller
> tmiller at newssun.med.miami.edu
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