M13 Phage Production Difficulties

[dni...@vax.oxford.ac.uk]

I am in Inorganic chemist who needs a few pointers on some basic
molecular biology techniques. I have transformed M13mp19 DNA containing the
insert of interest which I have mutagenised (I hope) into the mv1190 strain of
e-coli. IPTG/XGAL screening on LB plates shows many well isolated plaques, and
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.
My problem is that I have observed little or no phage after a PEG
precipitation which normally works with other growths we have done. The fact
that the plaques are forming on the plates at all seems to indicate (I think)
that phage is being packaged and released, and so I am baffled by my inability
to produce it in overnight growths.
An explanation I am considering atthe moment is a possible lack of
aeration (the tubes are only 15 ml, there is 5 ml of LB there, and a finite
amount of oxygen, athough they are shake vigorously) but I am not sure that
this would halt phage productioon like this. Is it possible thatthis may be my
problem? I am going to pick plaques and place the cocktail sticks in 5 ml of
LB in a 50 ml sealed tube tonight to see if that makes any difference.
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. Thank you in advance for any hints or
suggestions, please e-mail me directly as I do not check the net as often as I
would like (too much time in the lab trying to sequence dirty M13 DNA!! 8) )

Darren Nickerson
Inorganic Chemist Turned Mol. Biologist

[bu...@mbcf.stjude.org]

In article <1993May14.1...@vax.oxford.ac.uk>, dni...@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