Purification of Bacteriophages Using a Sucrose Density Gradient

Abstract

Source: Rustad, M., et.al. Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System. J. Vis. Exp. (2017)

This video demonstrates the purification of bacteriophages from a suspension containing host bacterial debris using a sucrose density gradient. It shows how the density gradient separates phages by buoyant density, enabling recovery of a visible band for resuspension and further analysis.

Protocol

1. Purification of Phage

NOTE: Sucrose purification is largely dependent on the size of the phage. Considerations for the mass of the phage to be isolated will have to be made, and adjustments to gradient conditions will be performed. Final viral titers of over 10¹³ phage/mL are easily achievable with this method.

1. Prepare 12 mL of 5% and 45% w/v sucrose in 1x TM buffer.
2. Prepare 4x 5 45% sucrose gradients by pipetting 2.5 mL of 45% sucrose into 4 ultracentrifuge tubes. Top with ~ 2.6 mL of 5% sucrose, stopping when the liquid reaches 2 mm from the rim of the tube.
   NOTE: Place the pipette tip into co....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ultracentrifugation tubes	Beckman Coulter	344057
Conical tubes	Falcon	352070
Gradient maker	BioComp Gradient Master	see Anal Biochem. 1985 Jul;148(1):254-9.
Syringe and Blunt Cannula	Monoject	8881513918 and 888202017