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Title
I) Purpose
- The purpose of this experiment is to grow T4 phage in the presence of 5’-bromodeoxyuridine, inducing mutations as the phage replicates.
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II) Expected Outcome
- This mutagenesis should produce some phage with multiple genomes packed inside of elongated capsids.
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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
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