High-Titer Phage Prep

SDB 9/96


1. Overnight culture

Inoculate 100 ml Super with colony of 7155 & bubble overnight at 30ºC


2. Subculture

Inoculate 1 L Super with 10 ml overnight culture & grow at 250 rpm, 30ºC


3. Infection

When cell density of cultures reaches 108/ml, add phage (m.o.i. = 0.1). Continue shaking at 250 rpm, 30ºC. Monitor cultures for lysis. Place lysed cultures on ice & add 0.5 ml chloroform. Six hours after infection place unlysed cultures on ice & add chloroform. Chill for 15 min.


4. Pellet debris

Transfer cultures to GSA bottles. Do not transfer chloroform. 10 min, 5K (GSA rotor), 4ºC


5. Precipitate phage

Decant supernatant into sterile 1 L plastic bottles (fill to about half an inch from neck). [If using phage next day for tailspike prep, prepare serial dilutions from supernatant before adding PEG & NaCl. Plate 0.1 ml of 106 dilution & 0.5 ml of 108 dilution.] If the culture lysed, also plate 0.1 ml of 108 dilution. Add PEG (7%) & NaCl (0.5 M). Completely dissolve PEG by inverting & swirling. Shake overnight at low speed in cold room.


6. Pellet phage

20 min, 8 K (GSA rotor), 4ºC


7. Resuspension & storage

Decant supernatant into dirty 1-L bottles. Add 10-ml lysis buffer (20 mM MgCl2, 50 mM Tris (pH 7.6)) to each GSA bottle. Resuspend pellets by shaking at low speed in cold room. Rinse pellet off of walls of GSA bottles with Pasteur pipette. Pool pellets & rinse with 10-ml lysis buffer. Store at 4ºC.


8. Titer

Plate 0.1 & 0.5 ml of 1010 dilution