Team:BYU Provo/Notebook/LargePhage/Springexp/Period1/Exp/Team:BYU Provo/Phage Liquid Culture Mutagenesis

Overview

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May-June

July-August

September-October

The purpose of this experiment is to grow T4 phage in the presence of 5’-bromodeoxyuridine, inducing mutations as the phage replicates.

II) Expected Outcome

This mutagenesis should produce some phage with multiple genomes packed inside of elongated capsids.

III) Materials

E. coli bacteria at density of 3.0 x 10^8 cfu/mL (OD600 of __)

Adenine

LB Broth (rich media)

5-bromodeoxyuridine

M9+ media (minimal media)

Chloroform

High titer phage lysate of T4 stock

IV) Methods

1. In a flask, start a culture of E. coli in rich media (LB) supplemented with 20 ug adenine per mL. Grow to a density of 3.0 x 10^8 cells/mL. (OD600 reading of ___)

2. Chill and pellet the E. coli. Then resuspend at a concentration of 1.5 x 10^8 cells/mL in M9+ medium supplemented with the following (per mL): 200ug 5-bromodeoxyuridine, 20 ug uracil, 20 ug adenine.

3. To a 30 mL amount of resuspended E. coli, add 2 x 10^9 phage particles and aerate until cleared.

4. Chill and pellet the bacteria, transfer to a new tube, and add chloroform to kill any remaining E. coli. Store at +4C.

V) Results