Method

1. Cool the lysed cultures containing bacteriophage A to room temperature. Add pancreatic DNase I and RNase, each to a final concentration of 1 pg/ml. Incubate the lysed cultures for 30 minutes at room temperature.

2. To each 500-ml culture, add 29.2 g of solid NaCl (final concentration, 1 M). Swirl the cultures until the salt has dissolved. Store the cultures for 1 hour on ice.

3. Remove debris by centrifugation at 11,000g (8300 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C. Combine the supernatants from the four cultures into a clean 2-liter graduated cylinder.

4. Measure the volume of the pooled supernatants and then transfer the preparation to a clean 2-liter flask. Add solid PEG 8000 to a final concentration of 10% w/v (i.e., 50 g per 500 ml of supernatant).

Dissolve the PEG by slow stirring on a magnetic stirrer at room temperature.

5. Transfer the solution to polypropylene centrifuge bottles, cool the bacteriophage/PEG solution in ice water, and store the centrifuge bottles for at least 1 hour on ice to allow the bacteriophage particles to precipitate.

6. Recover the precipitated bacteriophage particles by centrifugation at 11,000g (8300 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C. Discard the supernatants, and stand the inverted centrifuge bottles in a tilted position for 5 minutes to allow the remaining fluid to drain away from the pellet. Remove any residual fluid with a pipette.

7. Use a wide-bore pipette equipped with a rubber bulb to resuspend the bacteriophage pellet gently in SM (8 ml for each 500 ml of supernatant from Step 3). Place the centrifuge bottles on their sides at room temperature for 1 hour so that the SM covers and soaks the pellets.

8. Extract the PEG and cell debris from the bacteriophage suspension by adding an equal volume of chloroform. Vortex the mixture gently for 30 seconds. Separate the organic and aqueous phases by centrifugation at 3000g (4300 rpm in a Sorvall GSA rotor) for 15 minutes at 4°C. Recover the aqueous phase, which contains the bacteriophage particles.

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