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Amp satellites

Steve Minchin S.D.Minchin at bham.ac.uk
Thu Jul 14 09:32:48 EST 1994

In article <1994Jul11.172458.176 at immunex.com> gayler at immunex.com writes:
>From: gayler at immunex.com
>Newsgroups: bionet.molbio.methds-reagnts
>Subject: Amp satellites
>Message-ID: <1994Jul11.172458.176 at immunex.com>
>Date: 11 Jul 94 17:24:58 PST
>Organization: Immunex Corporation, Seattle, WA
>Lines: 28

>Here is a puzzler. We have recently had a problem with 
>satellite colonies on O/N growth of our Amp plates. This
>usually happens once a year or so. We have performed all 
>manner of controls, such as increasing Amp concentration 
>to 350ug/ml, making new Amp stocks, buying new Amp from 
>other vendors. We have spread Amp on top of fresh LB
>plates (15cm) at varying concentrations (up to the 
>equivalent of 1 mg/ml!) We still get some satellites, 
>particularly with high copy number plasmids. We see 
>satellites whether there are 200 colonies or if there are
>10.The number of satellites decreases with increasing Amp 
>but with certain plasmids, such a pGEX, they still remain. 
>We don't believe the Amp is being heat-inactivated since 
>we have taken special care, and the spreading experiments 
>used fresh Amp. Different people have also been involved 
>in making plates.We are using concentrations of Amp that
>It appears to me that something is inactivating the Amp but 
>I don't know what.
>We are getting somewhat frustrated. The problem will 
>probably go away soon, but I'm sure it will return again.
>Is there something that could be sequestering the Amp? Would 
>carbenicillin be more effective? Any help would be appreciated.
>                Thanks,
>                Richard Gayle
>                gayler at immunex.com
>The opinions expressed are solely my own. 

We have had similar problem. The solution which works most of the time is to 
reduce the grow out time before plating to between 30-45 minutes. 
Alternatively we (i) use 200ug/ml Amp + 200ug/ml Carb.
or  (ii) incubate the plates at 30 degrees C.

All the best

Steve Minchin
Dr. Steve Minchin,

School of Biochemistry, The University of Birmingham,
Edgbaston, Birmingham, B15 2TT, U.K.

InterNet:       S.D.Minchin at bham.ac.uk
Janet:          S.D.Minchin at uk.ac.bham


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