Bacteriophage precipitation using PEG (PEG/NaCl method)

Purpose

Concentrate bacteriophages from a cleared lysate using PEG (polyethylene glycol) + NaCl, then resuspend as a smaller-volume, higher-titer phage prep.

Safety

Work with the appropriate biosafety level for your host bacterium and sample source. Use PPE and disinfect surfaces (e.g., 10% bleach followed by ethanol).

Materials

• Cleared phage lysate (plate lysate or liquid lysate)
• NaCl (to 1.0 M final)
• PEG 8000 (typical final 10% w/v)
  (PEG 6000 can also work; PEG 8000 is most common)
• Sterile SM buffer or PBS (for resuspension)
  • Classic SM: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 8 mM MgSO₄, 0.01% gelatin (optional)
• Centrifuge tubes (50 mL, 250 mL, etc.)
• Centrifuge (4 °C preferred)
• 0.22 µm filter (optional but recommended)
• Gentle rocker/rotator

Before you start (key notes)

• Start with a well-cleared lysate: remove cells and debris first, or PEG will co-precipitate junk.
• PEG precipitation concentrates phage, but may also bring down proteins/vesicles—downstream cleanup (chloroform extraction, DNase/RNase, CsCl, ultrafiltration) is optional depending on your goal.

Step 1 — Clarify the lysate

1. Centrifuge lysate 10,000 × g, 10–15 min, 4 °C.
2. Transfer supernatant to a clean tube.
3. (Recommended) Filter through 0.22 µm to remove remaining bacteria.

Step 2 — Add NaCl (1.0 M final)

1. Add NaCl to 1.0 M final.
   • Example: for 100 mL lysate, add ~5.84 g NaCl (since 1.0 M NaCl ≈ is 58.44 g/L).
2. Mix until fully dissolved.
3. Incubate on ice or at 4 °C for 30–60 min.
4. Centrifuge 10,000 × g, 10 min, 4 °C to pellet precipitated proteins/debris.
5. Transfer the supernatant to a new tube.

Step 3 — Add PEG 8000 (10% w/v final)

1. Add PEG 8000 to 10% (w/v) final.
   • Example: for 100 mL lysate, add 10 g PEG 8000.
2. Mix thoroughly (PEG dissolves slowly; stir/rock at room temp briefly if needed, then cool).
3. Incubate at 4 °C for 2 hours to overnight (overnight usually gives the best yield).

Step 4 — Pellet the phages

1. Centrifuge 10,000–12,000 × g, 20–40 min, 4 °C.
2. Carefully decant the supernatant (PEG-containing) without disturbing the pellet.
   • The pellet can be transparent/whitish and slippery—easy to lose.

(Optional wash)
3. Add 1–2 mL cold SM along the wall, gently swirl, and remove to reduce PEG carryover.

Step 5 — Resuspend the phage pellet

1. Resuspend pellet in SM buffer/PBS:
   • Typical: 1/50 to 1/100 of the starting volume
     (e.g., 100 mL → resuspend in 1–2 mL).
2. Let sit at 4 °C for 30–60 min (or gentle rocking) until fully dissolved.
3. Optional: brief spin 5,000 × g, 5 min to remove insoluble material; keep supernatant.

Step 6 — Storage

• Short-term: 4 °C (days–weeks depending on phage)
• Long-term: -80 °C with cryoprotectant (commonly 10–20% glycerol), or store as lysate at 4 °C if stable.
• Avoid repeated freeze–thaws.

Quality checks (recommended)

• Titer before and after (plaque assay) to estimate recovery.
• If you need cleaner prep:
  • Dialysis (SM buffer) to remove PEG/salt
  • Ultrafiltration (e.g., 100 kDa cutoff)
  • Chloroform extraction (some phages are chloroform-sensitive—test first)

Troubleshooting

• Low recovery: incubate longer with PEG (overnight), ensure PEG and NaCl reach final concentrations, keep everything cold, and avoid losing the pellet.
• Pellet won’t dissolve: add more SM, resuspend gently, let sit longer at 4 °C with rocking.
• Downstream inhibition (PCR/enzymes): remove PEG by dialysis/ultrafiltration; PEG carryover is common.
• Phage inactivation: some phages don’t like high salt/PEG or long cold incubations—reduce incubation time or try milder PEG % (8%).

After precipitation next stage is usually centrifugation under Ceasium chloride (CsCl) gradient