Small-Scale Preparation of Bacteriophage by PEG Precipitation

Small-Scale Preparation of Filamentous Bacteriophage by PEG Precipitation

This procedure describes the small-scale preparation of filamentous bacteriophage by single PEG-precipitation. This protocol is used to prepare single phage and usually gives enough virions for routine binding analyses.

Material & Reagents

The following materials and reagents are needed for the procedure:

• A bench microcentrifuge, sterile DNase-free microtubes, aerosol-barrier tips, and a bucket of ice.

• TBS (1x) or Tris Buffer Saline: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, sterile, made 1x from commercial 10x stock solution(prevent possible protease contaminations).

• PEG/NaCl (5x) stock solution: PEG-8000 20%, NaCl 2.5 M. Dissolve 100 g PEG-8000 (20% w/v) and 75 g NaCl (2.5 M) in 400 ml ddH₂0 and bring to a final volume of 500 ml by stirring at RT. The solution can be autoclaved (optional) but mixing during the cooling period is required to prevent a phase separation. Store at RT.

Procedure

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

2. Spin down bacteria by microcentrifugation for 2 min at 13,000 g.

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

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

5. 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.

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

7. 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.

8. 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.

9. Vortex vigorously again and clear the phage solution by microcentrifugation for 1 min at 13,000 g; transfer the
   phage solution to a clean microtube and proceed to virion quantification.

Troubleshooting

Low virion counts

If your phage yields are too low, e.g. less than 0.1 O.D.x mL in the original culture, most likely virions are embedded with bacterial debris, the U.V. spectrum will exhibit a shallower minimum or worst, no minimum below 269 nm, and the concentration of virions measured by this method will be erroneous. It is technically difficult to purify phage further by PEG-precipitation in this situation.

To improve phage yields and take full advantage of this technique, 1) if you are using a helper phage, make sure that the density of the bacterial culture is optimal at the time of superinfection (e.g. around 0.4-0.5 OD600 for TG1 and SS320), 2) if you are using M13KO7 as a helper phage, switch to a more efficient packager such as CM13, and 3) more importantly, try to switch to a phage display system like the pADL Phagemid Vector System where expression is under tight control. Many old phagemid systems such as pComb3 or Phen2 vectors use a very leaky lac promoter driving overexpression of the fusion protein even in absence of IPTG, resulting in bacterial toxicity, slow bacterial growth, bacterial lysis and limited phage production.

Centrifugation Table

The protocol can be easily adapted to larger scale preparations by adjusting the centrifugation time while keeping the relative centrifugal force to 13,000 g as indicated in the following table:

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