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titer determination of phage M13K07

ijiwaru at wheel.dcn.davis.ca.us ijiwaru at wheel.dcn.davis.ca.us
Mon Jun 9 13:06:36 EST 1997

In article <tyr-2-0706971255440001 at news.srv.ualberta.ca>,
tyr-2 at bones.biochem.ualberta.ca (Karl Fischer) wrote:

> In article <hgtsei-0706971555000001 at ukmhg03.med-hg.uni-sb.de>,
> hgtsei at med-rz.uni-sb.de (Thomas Seib) wrote:
> <snip>
> Hello Thomas,
> I had similar problems with plaquing when I first started (though I was
> not working with phage display). A few things I found were important were:
> 1) The selection of host. 
> I started using an aliquot of JM109 which came with a kit and found that
> infection with R408 or K07 was pretty pathetic; I'm biased against JM109
> 'cause in the past it has always been a very sickly bug to work with.
> I switched to JM103 and things improved markedly.
> 2) The growth of host.
> I found that I could get consistent plaque production if I grew a colony
> of JM103 overnight in 5ml of M9 medium (with CaCl2 and MgSO4)-0.6%
> glycerol-0.2% casamino acids-0.002% thiamine at 37C. 
> 3) Infection of host.
> I make dilutions of phage in YT broth and incubate 100 ul of phage with
> 100 ul of JM103 (from above) at 37C for 15-30 minutes. Mix GENTLY by
> swirling; the F pilus is the point of attachment for these phages and
> rough treatment (eg. vortexing) can shear off the F pilus and *POOF*
> no/poor infection.
> I add an additional 100 ul of JM103 just prior to addition of YT top agar
> to ensure good lawn formation.
> Add YT top agar only *after* it has cooled to less than 40C; addition of
> too-hot top agar results in poor plaque formation.
> Using the above have resulted in consistent plaque formation following
> incubation of the plates at 37C for 16-20 hours.
> Hope this was of some assistance to you.
> Cheers
> Karl the hepB guy
> -- 
> Karl Fischer
> tyr-2 at bones.biochem.ualberta.ca

Just to add to Karl's suggestion, we have found it possible to select for
tetR, F- XL1-Blue by repeated passage in LB-tet.  Just to be safe, we
passage through minimal medium whenever we need to be sure that the F' is
still present.  The problem is the tetR is carried on Tn10 and could make
the jump.

Lyle Najita
Plant Pathology
University of California - Davis

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