rcaspi at sdcc3.ucsd.edu (Ron Caspi) wrote:
>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?
>Thanks for any input
I would surmise that either your ampicillin has gone south or that you
are still pouring your plates too hot, more than likely the later.
We pour at the same concentration you do but we cool or LB agar to 65
deg C and then wait until its cool enough to manipulate with our hands
before adding the ampicillin. We still experience satellites on our
plates but not generally for quite a period after plating... days to
weeks, and this appears to be vector dependent as well .
--Rover The Mad Molecular Biologist
rover at livnet.combarlow at picard.evms.edu