Oneill Lab:Lambda

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

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Working with bacteriophage &lambda;

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&deg;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 &asymp; 0.5
 * 4) *Resuspended cells can be stored at 4&deg;C and will last about 2 days

Plating Lambda
High titer phage stock can be kept at 4&deg;C for about 6 months.


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