Dr. Duncan Clark Duncan at genesys.demon.co.uk
Thu Jul 15 13:58:25 EST 1993

In article <1993Jul15.053557.28336 at gserv1.dl.ac.uk> vernon at micro.uct.ac.za writes:

>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
>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

Every protocol I have ever seen for T7 phage uses E.coli B or B derivatives.
I can't see any reason why HB101 won't work although it is a K12/B cross.
Maybe your phage stock is dead!

 Duncan Clark                       | Internet:   duncan at genesys@demon.co.uk
 GeneSys Ltd.                       | Compuserve: 100015.1406 at compuserve.com

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