Team:LCG-UNAM-Mexico/Resources/Protocols/P4

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=Rich Calendar's P4 vir1 growth protocol (and fun facts)=

This sheet was gently handed by Dr. Calendar when we cried for help with the Growth of P4 

P4 vir1 (11,627 bp; Nucl. Acids Res. 18:1649, 1990) is recommended as a length standard for pulse-field gel electrophoresis.

C-1895 F+ supD (P2 lg) is good for growing stocks of P4 and is a reasonable indicator for P4, because it gives large plaques if one pre-incubates cells and phage for 10 min at 37°C in the presence of 1 mM CaCl2 before plating.

C-2423 (P2 lg del1) trp-amber arg-amber rpsL(Str-r) is an even better indicator for P4. Grow the assay culture overnight in LB at 37° without aeration. Pre-incubate 0.35 ml cells and phage for 10 min at 37°C in the presence of 1 mM CaCl2. Add 2.5 ml LB soft agar and spread on an LB plate. Incubate at 37°C for about 8 hours. P4 does not make plaques at 30°C.

Starting from a single colony, grow a ten ml culture of C-1895 overnight (5PM-9AM) at 37° in Luria broth (0.5% NaCl) without aeration. To this culture add one freshly-picked plaque of P4 vir1, plus CaCl2 (1 mM) to promote adsorption. Incubate at 37° without aeration for 10 min and then add the infected culture to a 2800 ml Fernbach flask (which has a large opening for maximal aeration) that contains 400 ml LB broth supplemented with 0.1% glucose, 1.6 mM MgCl2 and 0.5 mM CaCl2. Shake at 37°, back and forth, 65 reciprocations/min. There should be waves rolling across the flask in order to assure good aeration. Start reading the A600 after 90 minutes. Lysis should begin at about 3 hours, when the culture has an A600 between 0.7 and 1.5. When lysis begins add 8 ml 4% EGTA (pH 8.8) to chelate the calcium ions and block phage re-adsorption. Also add 2.5 ml 1 M MgCl2 to stabilize the phage. When lysis is complete, remove the cellular debris by centrifugation in a GSA rotor at 5,000 rpm for 10 min. Then add 22.5 ml 1M MgCl2 to further stabilize the phage. One can expect 10^10 plaque-forming units/ml. There are two means to concentrate the phage:

1. Centrifuge in an SS-34 rotor at 13,000 rpm for 3 hours or for 2 hours at 15,000 rpm. Discard the supernatant. Drain tubes on a paper towel. Add 0.25 ml of P4 buffer to each tube, and allow the phage to re-suspend overnight in the refrigerator. P4 buffer is 0.08 M MgCl2, 0.01 M tris.Cl (pH 7.5), 1% NH4C2H3O2. Remove debris by centrifugation.

2. Add NaCl to 2.5% final concentration, and PEG-6000 to 8%. After the PEG dissolves, centrifuge as described above for removal of cell debris, and drain the pellet well by centrifuging very briefly a second time. Re-suspend the pellet in 4 ml P4 buffer. Add 2 ml CHCl3 and roll gently by hand to make an emulsion. Centrifuge at 8,000 rpm for 5 min to separate the phases. Remove the aqueous (upper) layer and measure its volume. This step removes PEG and other junk from the phage preparation.

If you require further purification, add CsCl equal to half the weight of solution and centrifuge in the SW50.1 rotor at 40,000 rpm for 36-48 hr. Remove the phage band with a syringe and needle, and dialyze P4 buffer. Phage purified this was loses some of the Psu protein, which stabilizes the phage capsid.

Rich Calendar

June 7, 2009