cloning confusion - HELP!!
bhjelle at vaxine.unm.edu
Tue Nov 19 18:50:26 EST 1991
In article <1991Nov18.230030.26660 at usenet.ins.cwru.edu> djt2 at po.CWRU.Edu (Dennis J. Templeton) writes:
>General rule: any colony that grows on an Amp plate but not in liquid
>culture IS A BOGUS COLONY!
I'll second that.
>Ok, so what are these bogus colonies???
>First guess-- your amp plates are old or have a low level of Amp in them
>for another reason (you DO let the agar cool before adding it, don't you?)
>More commonly, the problem has been that the colonies are not E.coli!
>may not be partially amp-insensitive. The most frequent culprit (I've seen
>it a half-dozen times) is some kind of yeast... (don't know the name) that
I haven't seen this, but I have seen several people attempt to grow up
"satellite" colonies on amp plates. These are colonies that are composed
of amp-sensitive bugs that are able to grow because they are near a
bona-fide amp-resistant colony that is spewing out beta-lactamase from
the periplasmic space.
To prevent this, just do your plating late in the afternoon and rescue
the plate promptly in the morning & put it at 4C. That will keep the
amp-resistant colonies from getting too large and generating satellites.
I would guess that nearly 100% of the time I have seen colonies that
don't grow in LB amp media, it is due to this mistake.
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