T7 PHAGE

[vernon]

Dear Netters,

I am posting the following message on behalf of a
colleague.  I will forward any replies to her.

Vernon Coyne
vernon at micro.uct.ac.za
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I was sent about 100 ul of T7 phage in approx. 100 ul Luria broth.
However, I have not been able to propagate phage from this
suspension.  I have tried adding approx. 10 ul of the phage
suspension to plating cells in 10 ml Luria broth and growing for 5 -
6 hrs, adding a few drops of chloroform, and then centrifuging to
pellet out the bacteria.  I have then added 100 ul of this 'phage
stock' to 100 ul plating cells, incubated for 20 min at 37oC, and
subsequently added this mixture to 4 ml soft Luria agar at 47oC which
was poured onto Luria agar plates.  I get no plaque formation at
all.  I have tried using E. coli HB101 and a F'- CSH strain as
plating bacteria.  I have also tried growing a culture of HB101,
spreading 100 ul on a Luria agar plate, and spotting 10 ul of the
original T7 phage I was sent onto this in the hope that I would get a
maxi plaque.

I would like to know if I am using the correct strains for T7 phage
propagation, if my methodology is correct, and if there is something
essential that I need to include in the media; ie. maltose in the case
of lambda phage.

Any assistance in this matter would be appreciated.
Ros Powles