pET and ampicillin

[jim hartley]

	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.

Jim Hartley
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
> 
> 
>