Dr. Duncan Clark
drc at nospam.demon.co.uk
Thu Jul 27 07:47:47 EST 2000
In article <8lp6e0$4dm$1 at lux1.biobase.dk>, Jesper S. Pedersen
<jsp at NOSPAMimage.dk> writes
>I use a special kind of Helper Phage, but either it is mutating or I have a
>contamination of another kind of helper phage. I grow my Tg1 to an OD of
>about 0,5 and infect with my phage repertoire. When I count the cfu (using
>dilution series) on ampicilin and canamycin plates I get around the same
>amount - e.g. : 5e12 on ampicilin and 3e12 on canamycin. This could indicate
>that around half of the plasmids rescued are (helper-)phagemids.
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.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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