P1 transduction problems

"", David Alexander dalex at NEXUS.MICROIMM.MCGILL.CA
Wed Feb 17 00:27:08 EST 1999


Hello Netters,

I am attempting to transduce a non-essential gene containing
a TetR marker from E. coli MG1655 to MC4100.
The P1 vir lysate made on the MG1655 strain with my gene/TetR
is of very high titer (believe it or not, around 10 ^ 16)
However, I have yet to see a TetR transductant.

I am using the tried and true procedure from Miller:
5 mL over night of MC4100 + 5 mL MC Buffer (Mg, Ca)
incubate 37C for 15 min with shaking
mix 0.1 mL cells with 0.1 mL diluted phage lysate from MG1655
incubate at 37C for 20 min to let phage attach
then, add 0.2 mL 1M NaCitrate and plate onto LB + Tet.

The biosci archives had some helpful hints, but
to the best of my knowledge, MC4100 is RecA+
and I am using low levels of Tet in my LB plates.

Initially I thought that my multiplicity of infection was too high
(i.e. too many phage - no surviving E. coli)
but I have attempted the transduction over a wide range 
of phage concentrations (10 ^-2 through 10^ -24) 
and still no TetR clones.

I'm anxious to create this strain 
and would appreciate any suggestions.

Thank you,

David

dalex at microimm.mcgill.ca



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