In Article <1994Jul11.172458.176 at immunex.com>, gayler at immunex.com wrote:
>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.
> Richard Gayle
>gayler at immunex.com>--
>The opinions expressed are solely my own.
First, if you use carbenicillin, you should have less of a problem, as this
antibiotic is inactivated more slowly than AMP. Secondly, what host strains
do you use, and what medium. Media with glucose will allow the formation of
satellites. Thirdly, if the competent cells you use have been downshifted
from rich medium just prior to making them competent, this problem will
occur quite frequently. Hope this helps some.
Tracy Aquilla, Post-doctoral Research Associate
Department of Molecular Physiology and Biophysics
University of Vermont, College of Medicine
aquilla at salus.med.uvm.edu