supF plasmid libraries and growth of MC1061/p3 or DH10B/p3 bacteria

Bernard P. Murray, PhD bpmurray*STUFFER* at
Sun Nov 22 16:31:03 EST 1998

In article <l03110700b27df287ecae@[]>,
mtw3p at AVERY.MED.VIRGINIA.EDU (Mark Worthington) wrote:

> I have received libraries in pCDM8, a supF containing plasmid through which
> antibiotic selection is mediated through complementation of an amber mutant
> in the AMP and TET resistance genes on the p3 episome.  In two strains
> containing this episome (MC1061/p3 and TOP10/p3), the colonies come up
> fairly late (some at 16 hours, more at 36 hours) and, as a result, there is
> a range of colony sizes.  The concentration of Amp and Tet in the plate
> makes no difference, as far as I can tell.
> Is this a normal feature of this plasmid?  Any thoughts about when to
> harvest the plates?
> Thanks in advance for your assistance.
> Mark Worthington
> University of Virginia

I assume that you are using the double antibiotic selection
(7.5 - 10 mg/l tet and 25 - 40 mg/l amp) and if you want to
ensure that you have the P3 you can include kan.
     There is an appreciable spontaneous reversion associated
with the amber mutants so you *have* to do the double
selection and you shouldn't use the "normal" levels of
tet and amp (eg. 25 and 100 mg/l, respectively) or you
will be selecting for these revertants.  Make sure the
tet is fairly fresh and that you ar not using a medium
with Mg2+.
     For this reason I'd also suggest that you pick the
earliest decent colonies.
     Good luck,
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA

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