Preparation of Bacteriophage Lysate for Generalized Transduction

Abstract

Source: Saragliadis, A., et al. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. J. Vis. Exp.(2018)

This video demonstrates preparation of a bacteriophage lysate containing transducing particles. A donor strain is infected, plaques form, and the soft agar overlay is processed with chloroform to yield a clarified lysate for downstream analysis.

Protocol

1. Infection of the donor strain
   1. Grow the donor strain JW4197 in 5 mL of lysogeny broth (LB) medium supplemented with 10 mM CaCl₂ and optionally with Kan (25 µg/mL) to an optical density at 600 nm (OD₆₀₀) of ~1.0. Measure the OD₆₀₀ value using a spectrophotometer.
   2. Make a dilution series of an existing P1 phage stock in the LB medium: recommended dilutions are between 10^-3 to 10^-7.
   3. Mix 200 μL of the bacterial suspension and 100 μL of a given phage dilution in a 15 mL centrifuge tube or equivalent. Prepare as many tubes as phage dilutions. Incuba....

Results

Figure 1: Examples of plates after a P1 infection. (A) This panel shows a plate with individual plaques. (B) This panel shows a semi-confluent plate. (C) This panel shows an over-infected plate.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Strains
E. coli JW4179	NIG	JW4179-KC	tamA deletion mutant
P1 vir	NIG	HR16	Generally transducing bacteriophage
Agar	BD Bacto	214010
Calcium chloride	Merck	102382
Chloroform	Merck	102445