M13 Phage Production Difficulties

bugg at mbcf.stjude.org bugg at mbcf.stjude.org
Thu May 20 14:51:17 EST 1993


In article <1993May14.154329.14035 at vax.oxford.ac.uk>, dnicker at vax.oxford.ac.uk writes:
> 
  ...
> I have picked 10 of these plaques and innoculated them into 5 ml of LB in a 15
> ml screw-top tube.  I grew these overnight in the hopes of producing enough
> phage in the supernatant to prepare stocks, then planned to continue witha mini
> plasmid prep to confirm the mutation by sequencing> 	I would appreciate some advice here, as though this seems to be a basic
> point I have not been able to produce viable phage stocks with which to
> re-infect the larger growths I require....
> 
You say that you inoculate the plaques into 5 ml of LB.  If there are no cells
already growing in the LB, you may have trouble ever getting enough phage to
work with.  What we (and most people!) do is grow up a 50 ml culture of the
E.coli host by inoculating it 1:200 with an overnight culture, or even just a
loopful from a plate.  Put the 50 ml into something large like a 500 ml baffled
shake flask.  Let this culture grow with vigorous shaking for 1 hour.  Then,
use 5 ml of this culture in each of your tubes and add a plaque with a
toothpick or Pasteur or whatever.  If you are using screw-cap tubes, put
the tops on loosely and tape them on so they won't fall off.  Then, only grow
this culture for about 5 hours also with vigorous shaking - any longer 
is supposed to cause trouble with recombination or something!  I do not use 
PEG precipitation anymore, I just directly extract the culture with Tris-
saturated phenol.  The single-stranded DNA that results is excellent for 
sequencing.  If you would like the protocol for this, let me know.

Barbara Bugg
Molecular Pharmacology
St. Jude Children's Research Hospital
Memphis, TN  USA  38105


							Darren Nickerson
> 					Inorganic Chemist Turned Mol. Biologist



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