CHP - last updated: January 21, 1999
(1) innoculate 500 ml super with 5 ml 7155 (su+) overnight culture 1/100 dilution
(2) grow at 30oC to 1x108 cells/ml - mid log cells should look like sausages
(3) infect with phage of choice with MOI of 0.07-0.1
volume of phage stock added = (1x108) (0.07)(vol. ml)/stock titer
Take a sample of cells and plate for phage contamination
(4) continue to culture for up to 6 hours, multiple round of infection will occur till all the cells are infected and a clearing of the culture occurs.
(5) check for lysis:
(6) continue to culture for ~30 minutes after cells look lysed/lysable
(7) chill on ice, add 3 ml CHCl3, wait a few minutes to see the amount of phage you have, 6.1 6.5 & 8.5
(8) pour into GSA bottles - leave CHCl3 behind it will pellet first.
(9) spin 5K for 10 minutes, pellet debris
(10) pour supernatant into 1L erlynmeyer (pellets should be small) [can titer supernatant at this point to calculate final volume better]
(11) To precipitate phage, stir supernatant in 4oC
room, bring to: 7% PEG (6-8K), 0.5M NaCl
(for 500 ml: 35 g PEG, 14.61 g NaCl)
(12) let stir at least 1 hr or overnight in cold
(13) spin suspension at 8K rpm for 20 minutes, to bring down phage.
(14) resuspend pellet in 50 mM Tris pH 7.6, 100 mM MgCl2 (phage buffer), 25 ml volume
(15) Plate 10 10 0.1 10 10 0.5, calculate titer