Team:LCG-UNAM-Mexico/Resources/Protocols/P2

=Rich Calendar's P2 vir1 growth protocol=

This protocol was gently handed to us by Richard Calendar when we were crying for help in phage use

Plaques were made on LB plates using E. coli C-520 as indicator. I plaque was picked with a Pasteur pipette. For liquid growth of P2 vir1, a 50 ml LB culture of C-520 in a 500 ml flask was grown overnight at 37°C without shaking. Its A550 was 0.88. I plaque was pre-adsorbed to 1 ml of culture with 5 mM CaCl2 for 15 min at 30°C. The pre-adsorbed cells, plus 25 ml more of cells were added to 400 ml of LB supplemented with 0.5 mM CaCl2, 1.6 mM MgCl2 and 0.1% glucose. The mixture was shaken at 37°C in a 2800 ml Fernbach flask, so that waves rolled back and forth across the liquid. The A550 was 0.06 at the start. It increased to 0.95 over two hours and forty minutes. Five minutes later the A550 dropped to 0.92, and 8 ml of 4% EGTA were added to block phage re-adsorption. The A550 dropped to 0.2 over twenty minutes, and 2 ml CHCl3 were added for five minutes with shaking. The A550 dropped to 0.15. The culture was centrifuged in a GSA rotor at 6,000 rpm for at least 10 minutes to remove bacterial debris. The phages were centrifuged in an SS-34 rotor for 3 hours at 13,000 rpm or for 2 hours at 15,000 rpm. The supernatant was discarded, the tubes were drained on a paper towel, and the pellets were left overnight with 0.25 ml per tube of phage buffer, which is 10 mM tris-HCl pH 8.0, 10 mM CaCl2, 1% ammonium acetate. Remaining debris was removed by centrifugation in the SS-34 rotor for 10 minutes at 8,000 rpm. The phage were sterilized by filtration and stored in a tightly capped plastic tube in a dark refrigerator.

C-520 is E. coli C prototrophic F+ supD (Sunshine, M. G., M. Thorn, W. Gibbs, and R. Calendar. 1971. P2 phage amber mutants: characterization by use of a polarity suppressor. Virology 46:691-702).

P2 vir1 is a clear plaque-type mutant of P2 (Bertani, L. E. 1960. Host-dependent induction of phage mutants and lysogenization. Virology 12:553-569).

Rich Calendar

May 29, 2007