titer determination of phage M13K07

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Sat Jun 7 14:55:44 EST 1997


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





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