Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology
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
- Grow an overnight culture in LB supplemented with Mg2+ at 37°C shaking
- Pellet the cells at 2000 rcf for 5 minutes, and remove media
- 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
High titer phage stock can be kept at 4°C for about 6 months.
- Heat LB or NZY agar plates supplemented with Mg2+ to 37°C.
- 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.
- Add about 4 μL of a high titer phage stock to 200 μL resuspended cells.
- Incubate at 37°C for 15 minutes to allow phage to attach to cell walls.
- 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.
- Incubate at 37°C for 14-16 hours or until plaques grow to desired size