Editing Phage Purification (section)

== <br/>Procedure ==

Transfer 1500 µl of bacterial culture containing the phage particles to a microfuge tube.

<br/>Spin down bacteria by microcentrifugation for 2 min at 13,000 g.

Transfer 1200 µl of supernatant to a clean microfuge tube taking care of not touching the pellet of bacteria with the tip.

Add 300 µl of PEG/NaCl 5x and mix thoroughly by inversion; do not vortex.

Chill the tube on ice. After a few minutes, take the tube out; wipe it with a clean tissue and expose the supernatant to indirect lighting. By rocking the tube back and forth with the fingers, PEG-precipitated virions are often seen by the naked eyes. If a precipitate is visible, the incubation can be shortened to 5 min, otherwise continue the incubation on ice up to a full hour. Some protocols in the literature suggests just 5 min on ice. Note that long incubations may promote proteolysis, especially when epitope tags are present, and it is likely good practice to shorten the precipitation as much as possible.

Pellet the virions by microcentrifugation for 3 min at 13,000 g.

Carefully remove the bulk of supernatant with a large tip and discard in an appropriate container, taking care of not spreading bacteriophage on gloves and pipettors; microfuge again for 1 min at 13,000 g; remove all residual supernatant with a 100-µl tip and discard the tip. The second centrifugation is essential to 1) collect all the phage particles at the bottom of the tube and 2) achieve a complete removal of bacterial supernatant.

Resuspend the pellet by vigorous vortexing with 120 µl of TBS (1x) (1/10th of the initial culture volume) and incubate on ice for another hour. Sometimes, it is easier to let the pellet soften for a few min before resuspending the virions. Again, the incubation time can be shortened when large amounts of virions were precipitated but a safe 30-min incubation step is recommended to prevent a loss of particles during the clearing step.<br/>Vortex vigorously again and clear the phage solution by microcentrifugation for 1 min at 13,000 g; transfer the<br/>phage solution to a clean microtube and proceed to virion quantification.