Jesper S. Pedersen
jsp at NOSPAMimage.dk
Thu Jul 27 09:33:40 EST 2000
Dr. Duncan Clark <drc at nospam.demon.co.uk> wrote in message
news:vdL1ySAz9Cg5EAoC at lineone.net...
> If, as it seems, you are plating the helper rescued culture supernatant
> into an F' containing E.coli for the titre then you are seeing exactly
> what you should see. The kanamycin resistant helper will propagate
> itself in the F' E.coli so you will get kanamycin resistance.
> Titre everything onto a F' minus E.coli and then you should only pick up
> the Ampicillin phagemid. The helper phage will not grow.
> I think you will need to read a little more about the system you are
> working with and strain genotypes.
F' is necesary for phage infection, right? So how can you titre the phage on
F' minus E. coli? I think you misunderstood med : Under phage production it
seems that 50% of produced contain the wanted phagemid and 50% contain the
helper-phage genome. This seems to be a fact... The question is : How can
you efficiently 'kill' the bacteria that are infected with a phage
containing the helper-phage genome - to prevent them from producing new
I'm developing a new selection strategy - so there is nothing for me to read
at the moment - and no experts to ask :). The problem is that I can do one
round of selection perfectly rescuing with the correct helper phage, but
when doing the second round it seems that some helper phage transfered from
the first round of selection dominates the second round. To make my
selections work correct I need to prevent helper phage beeing transferred
from the first round to the second. Using a low titre of phage should insure
that only one (less than one) phage enters each bacteria. This is why I need
to know if a bacteria "stopped" by ampicilin would be completely unable to
produce phage. Another problem is that if both the helper-phagevector and
the phagemid in some rare cases are packaged into one phage particle the
bacteria infected with this phage would be able to still produce phage.
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