Purification of Bacteriophages from Bacterial Lysate

Resumo

Source: Pett, N., et al. T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo. J. Vis. Exp. (2024)

This video demonstrates the purification of bacteriophages from bacterial cell lysates using sequential centrifugation, membrane filtration, chloroform extraction, centrifugal filtration, and buffer exchange to obtain a purified phage preparation.

Protocolo

1. Preparation of experimental phage lysates
   1. Culture E. coli in 5 mL of LB medium in sterile polystyrene or glass culture tubes (with caps). Incubate tubes overnight at 37 °C with shaking at 200 rpm, until the stationary phase is reached.
   2. Subculture the overnight E. coli culture 1:50 in 100 mL LB medium in a glass conical flask. Incubate at 37 °C with shaking at 200 rpm until the bacteria have reached early to mid-exponential phase (approx. 1.5 h).
      NOTE: An initial growth curve can be performed to determine the optical density at 600 nm (OD₆₀₀) range at which the bac....

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
50 ml PES Steriflip Sterile Disposable Vacuum Filter Units	Millipore Sigma	SCGP00525
Amicon® 100kDa Ultra-15 centrifugal filter device, Ultracel-100	Millipore Sigma	UFC910008
BD Microtainer® Tubes, SST	BD Medical	365967
Chloroform (Ethanol as Preservative/Certified ACS)	Fisher	C298-500
MaxQ 6000 Incubated Shaker	Thermo Scientific	8354-30-0009
Microbiology Media: LB Broth (Powder) - Lennox	Fisher BioReagents	BP1427-500
Microcentrifuge Tubes with Locking Snap Cap, 2ml	Fisher	14-666-315
Phage stocks	Carolina Biological Supply	n/a