P1 transduction of Cm marker

Stephan Heeb StephanX.HeebX at lbm.unil.ch
Mon Mar 22 05:09:06 EST 1999


Hi all,

I'm attempting to transduce a Cm marker from one E. coli to another
(CSH50). I've done the stock (10e9 pfu/ml) and have attempted only once
the transdcuction, following a modified Miller protocol, without any
success.

The modification of the original protocol is the following:

  -After preadsorbtion of the phage and addition of 1M sodium citrate, 0.6
ml of  MME medium is added (final vol. 1.0 ml). The cells are then
incubated for 1 hour at 37°C with shaking, and then plated on MME plates
containing 20 ug/ml Cm.

The aim of the extra incubation time is to allow for the recombinations to
happen before plating on selective media. MME medium is used, since it
contains citrate to chelate calcium. We also don't want a lysogenic
result.

Before modifying this protocol and attempting again, I would like to know
if there's someone here that has some PRACTICAL EXPERIENCE with this
transduction that could give me some advice.

Thank you very much,

   Stephan Heeb

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