Oneill Lab:Lambda

Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

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Working with bacteriophage λ

Preparing Host Cells

Select an appropriate strain of E. Coli and adjust growth media as needed. For this protocol the strain is XL1 Blue MRA

1. Grow an overnight culture in LB supplemented with Mg²⁺ at 37°C shaking
2. Pellet the cells at 2000 rcf for 5 minutes, and remove media
3. Resuspend cells in 10 mM MgSO₄ to an OD₆₀₀ ≈ 0.5
   • Resuspended cells can be stored at 4°C and will last about 2 days

Plating Lambda

High titer phage stock can be kept at 4°C for about 6 months.

1. Heat LB or NZY agar plates supplemented with Mg²⁺ to 37°C.
2. Melt LB or NZY top agar supplemented with Mg²⁺ and keep at 50°C until ready to use.
   • Top agar is made by adding 0.7% agar to media
   • Melt top agar thoroughly. Small particles will give plates a rough surface.
3. Add about 4 μL of a high titer phage stock to 200 μL resuspended cells.
4. Incubate at 37°C for 15 minutes to allow phage to attach to cell walls.
5. Add cells to 3 ml top agar and plate immediately.
   • Top agar will solidify rapidly, tilt plate to spread evenly.
   • Be sure top agar has cooled to 50°C before adding cells.
6. Incubate at 37°C for 14-16 hours or until plaques grow to desired size