CHP - last updated: January 28, 1999
(1) - grow 30 ml 7155 (su+) in LB overnight
(2) - add 0.5 ml 7155 overnight or 0.5 ml. plating bacteria and 1 plaque to 30 ml LB media
- push all the way down into the plate
- one capillary tube obtains ~106 phage
(3) grow 3-6 hours at 30oC (or other more permissable temperature, e.g 20-25oC overnight) until lysis occurs
A fraction of the cells will be infected, the remaining cells will go through about 2 doubling. In the infected cells with lysis, the phage will infect a portion of the cells, the remaining cells will double; this will continue till essentially all the cells are infected and they lysis which will be noted by a clearing of the culture.
(4) add a few drops of CHCl3 to ensure complete lysis
(5) pour into ss34 centrifuge tube without pouring the CHCl3 into the tube
The CHCl3 will be the first to pellet so any debris that pellet on top will slide right off when you pour off the sup't. In addition the CHCl3 will eat the plastic.
(6) spin supernatant 15K 90 minutes to pellet phage and separate from lipopolysaccharides
It is the phage bacterial DNA complex that is pelleting; if you have purified phage you will need to pellet at 35K 45"
(7) discard supernatant and blot to remove residual LB
(8) pellet debris by spinning 10K 10 minutes and transfer into a tube with 1-2 drops of CHCl3 to prevent a further round of growth