Ampicillin plates

John W. Rooney rooney at mbcrr.harvard.edu
Fri Dec 8 19:18:36 EST 1995


Hi Michael:

		I use an alternative antibiotic called carbenicillin that eliminates 
satellite colonies.  You can buy it from Sigma.  It's a bit more 
expensive than amp.

Good Luck--John Rooney



-------------- next part --------------
From: abel at UTKVX.UTCC.UTK.EDU ("Michael Gregory Abel, University of Tennessee")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Ampicillin plates
Date: 7 Dec 1995 11:01:58 -0800
Organization: BIOSCI International Newsgroups for Molecular Biology
Message-ID: <Pine.PMDF.3.91.951207133222.543743680A-100000 at utkvx.utk.edu>
References: <jnakamot-0712950342240001 at ts36-8.wla.ts.ucla.edu>

> >
> > Lately we have been having hard time with ampicillin plates in our
> > lab. I can't figure out what we're doing wrong: we prepare stock
> > solution of 100 mg/ml ampicillin in water, and add them to LB medium
> > just before we pour the plates (not too hot) to a concentration of 100
> > or 150 ug/ml. Alas, there is a major problem of satelite colonies
> > growing around the resistant colonies when we spread-plate
> > transformation mixtures. Any idea?

> > Ron Caspi
> > Scripps Institution of Oceanography       Phone   (619) 534-0638
> > La Jolla  CA  92093-0202                  Fax     (619) 534-7313
> 
>This has become a problem in our lab, too, and I suspect it has something
> to do with the age of the ampicillin powder we keep in our refrigerator
> (how old is yours?). If anyone knows the suggested effective lifespan of
> ampicillin powder at +4 degrees C, please let me know. 
> 
> Jon Nakamoto, MD
> UCLA Medical Center
> jnakamot at ucla.edu

As far as I know, the shelf life of Ampicillin in a powder form 
maintained at 4oC in a relatively moisture free environment should be a 
couple of years (at least?).  In our lab, we make up ampicillin stocks at 
100mg/ml in 70% EtOH and store at -20oC.  The EtOH keeps the amp from 
freezing and the stocks last much longer than at the low temperature.  
Since the ampicillin is in a 1000X concentration, when it is added to 
your medium the EtOH volume becomes insignificantly small.  There is not 
a problem with repeated freeze/thaw because the ampicillin solution never 
freezes.  Also, the -20oC amp is very convenient to use because you don't 
have to wait for it to thaw, it doesn't need to be aliquoted, less of the 
powder is used due to a centralized stock available for the entire lab to 
use instead of everyone having their own personal stock.
	If you still have satelite colonies, you may want to add into 
your medium Methicillin (an ampicillin derivative antibiotic) at 80 to 
100ug/ml final concentration along with your ampicillin.  I'm not quite 
sure how it cuts down on satelite colony formation so I won't speculate 
(if anyone knows for sure and have a few minutes to bounce me the answer, 
I would like to know).

Hope this helps,

   ***********************************************************************
   Michael Gregory Abel    Abel at utkvx.utk.edu    423-974-2933 (office)
   University of Tennessee    Dept. of Microbiology    423-974-4007 (FAX)
   Knoxville, Tennessee 37996-0845    
   ***********************************************************************




More information about the Methods mailing list