Dec 17, 2022
Phage amplification and concentration
- 1Arcadia Science
Protocol Citation: Adair Borges, Januka Athukoralage 2022. Phage amplification and concentration. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmnb86g3p/v1
Manuscript citation:
Borges A, Radkov A, Thuy-Boun PS. (2022). A workflow to isolate phage DNA and identify nucleosides by HPLC and mass spectrometry. https://doi.org/10.57844/arcadia-1ey9-j808
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 21, 2022
Last Modified: January 29, 2024
Protocol Integer ID: 65060
Keywords: phage, gDNA, nucleoside, genome modification, HPLC, Mass spectrometry
Abstract
This protocol details methods to amplify bacteriophages T4 and SPO1, and concentrate using either PEG precipitation or filtration.
Phage amplification
Phage amplification
For each host, prepare a 30 mL subculture at 1:100 dilution (300 µL in 30 mL). Grow for 2 h at 37 °C in a shaking incubator. Subculture media (LB or ENB) should be supplemented with 1 mM CaCl2 and 1 mM MgCl2.
Grow a 3 mL overnight culture of phage host at 37 °C with shaking. Escherichia coli strain B (ATCC Strain 11303) should be propagated in Lysogeny Broth (ATCC Medium 1065), and Bacillus subtilis strain 168M (ATCC Strain 27370) is propagated in Enriched Nutrient broth (ATCC Medium 265).
Note
ATCC Medium 265: Enriched Nutrient Broth
Heart Infusion Broth (BD 238400) – 12.5 g
Nutrient Broth (BD 234000) – 5.4 g
Yeast Extract – 2.5 g
DI Water – 1000 ml
Autoclave at 121 °C.
Note
ATCC Medium 1065: LB Agar/Broth, Miller
LB, Miller Composition
Tryptone – 10.0 g
Yeast extract – 5.0 g
Sodium chloride – 10.0 g
*Agar – 15.0 g
Final pH 7.0 ± 0.2. Autoclave at 121 °C.
*Omit agar for broth medium.
Note
Note: In subsequent experiments we propagated Bacillus subtilis strain 168M in LB broth, and amplified phage SPO1 under identical conditions as Escherichia coli strain B and phage T4 with no noticeable difference in bacterial or phage growth.
Add ~107 PFU of phage T4 (ATCC Strain 11303-b4) to the E. coli subculture, and ~107 PFU of phage SPO1 (ATCC Strain 27370-b1) to the B. subtilis subculture. Return the infected subcultures to the incubator, at 37 °C with shaking. Grow until the culture is cleared (around 5 h). If the culture doesn't completely clear within 5 h, you can let the infection proceed overnight.
Phage isolation and concentration
Phage isolation and concentration
Move the phage lysate to a 50 mL conical tube, and add ~1 mL of chloroform. Seal the tube tightly, and shake on a platform shaker for 10–30 min. Transfer to a new conical tube. Then, spin the lysate down in a centrifuge at maximum speed for 30 minutes.
Carefully pipette off the supernatant from the spun-down culture into a new 50 mL conical tube, avoiding any debris at the chloroform interface. Also avoid the chloroform. Move to a new 50 mL conical tube, and spin again at max speed for another 30 min or until supernatant is clear. Move to a new 50 mL conical tube.
To concentrate the phage lysate, we have used both PEG precipitation and a filter-concentrator based protocol. The PEG protocol requires an overnight incubation, but the filter-concentrator generally requires more hands-on time. Use whichever phage concentration protocol works best for your circumstances.
Phage precipitation with PEG5 steps
PEG prep requires an overnight incubation step at 4 °C.
To PEG-precipitate phage, first prepare 5× PEG precipitation solution containing 2.5 M NaCl and 20% w/v PEG8000.
Note
5× PEG solution (500 mL)
5 M NaCl stock solution – 250 mL
PEG8000 – 100 g
DI water – to 500 mL
Stir until dissolved. Sterilize with with 0.22 µm filter. Store at room temperature.
Add PEG precipitation solution to the phage lysate to obtain a 1× concentration of 0.5 M NaCl and 4% w/v PEG8000. Mix by inverting the tube several times and refrigerate overnight at 4 °C.
After the overnight incubation with PEG precipitation solution at 4 °C, pellet the PEG precipitated phage particles by centrifuging at 19,000 × g for 60 min at 4 °C. A small white pellet should form at the bottom of the 50 mL conical tube.
Remove the supernatant, being careful not to disturb the PEG-precipitated phage pellet.
Resuspend the PEG-precipitated phage pellet in 300 µL SM buffer. Store at 4 °C.
Citations
Step 8
Bonilla N, Rojas MI, Netto Flores Cruz G, Hung SH, Rohwer F, Barr JJ. Phage on tap-a quick and efficient protocol for the preparation of bacteriophage laboratory stocks.
https://doi.org/10.7717/peerj.2261