khatipovNO at NOuchicago.edu
Fri Jun 7 14:29:48 EST 2002
Most probably you just picked the satellite colony (non-resistant = not
baring a plasmid) that grown after amp has broken down in the medium due to
overincubation. Amp is not very stable and will brake down at 37C over a few
days. Take the original plate, wash of all the cells from the surface and
re-streak (or better, spread) a portion on a fresh amp plate. Next day pick
the resistant colonies. Otherwise, you may try to pick the largest colonies
from the original plate: most probably these are (were!) the
"Paul Wary" <paul_wary at yahoo.com> wrote in message
news:bvv1gusocskj2imrk6a47qiokru5ihv4gi at 4ax.com...
> Can bacteria that were streaked out on agar plates containing
> ampicilline lose their transformed plasmid (containing AmpR) if they
> have been incubated at 37 °C for several days (instead of being
> incubated for 1-2 days only and then being stored at 4 °C)?
> Is there any chance that bacteria which have lost the plasmid
> (containing AmpR) can still grow to some degree in LB broth media
> containing ampicilline?
> I ask these question because in an experiment of mine I did just what
> I described above and after growing up cultures from the
> "over-incubated" streaks (Note: The cultures were growing unusually
> slowly.), I tried to isolate plasmid DNA and the yield was extremely
> low. I checked all possible errors regarding the plasmid isolation
> procedure but couldn't find anything. So, I figured my bacteria might
> have lost the plasmid.
> I'm thankful for any comments.
> Paul W.
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