Moderate Scale Lambda Phage DNA Prep

Schedule:

From a single plaque in 1 ml SM + 30 µl CHCl₃, take 50 µl and add to 100 µl of bacteria.
- Assume that 1 plaque contains 10⁶ phage particles. This means you are preadsorbing 50,000 phage.
- To prepare bacteria for plating grow the bacteria in TMM to an A₆₀₀ of no more than 1, pellet the cells and resuspend in 10 mM MgSO₄ to an A₆₀₀ of 2.
Incubate at 37° C for 30 minutes
Mix phage with 3 ml of NZY Top Agarose (melted and cooled to 50°). Pour onto a small NZY plate.
Incubate at 37° overnight.
The plate should be be a lawn of plaques. Overlay with 3 ml of SM and shake gently for at least 2 hours to elute the phage particles.
Transfer 1 ml of eluate to a 1.5 ml Epindorf tube.
Add 50 µl of CHCl₃ and vortex briefly.
Spin in a microfuge for 10 minutes and transfer the aqueous fraction to a new 1.5 ml Epindorf tube.
Add 30 µl of CHCl₃. The titer of the phage should be between 10¹⁰ and 5 x 10¹⁰ pfu per ml.

Mix 10⁶ phage particles with 500 µl of bacteria (A₆₀₀ = 1 in 10 mM MgSO₄). Incubate at 37° for 30 minutes.
Add the preadsorbed phage to 37 ml of NZY in a 125 ml Erlenmeyer flask.
Incubate at 37° with vigorous shaking for approximately 5 hours.
When the bacteria have lysed (The culture should first get cloudy and then clear with cell debris visible.), transfer the entire culture to an Oakridge tube containing 100 µl CHCl₃.
Vortex the Oakridge tube and add 4 µl of a 10 mg/ml DNase I stock and 15 l of a 10 mg/ml RNase A stock. Incubate at 37° for 30 minutes.
Add 2.1 g of NaCl and mix gently to dissolve. Place the tube on ice for 5 minutes.
Centrifuge at 4° for 20 minutes at 7000 rpm in the Sorval using the SS-34 rotor.
Pipet the supernatant to a new Oakridge tube that contains 3.7 g of PEG 8000.
Dissolve the PEG with gentle shaking at 37° to 45°.
Incubate on ice overnight. You can shorten this step to as little as 1 hour, if necessary.
Centrifuge at 4° for 25 minutes at 7000 rpm in the Sorval using the HS-4 rotor.
Decant the supernatant and drain the pellet well.
Gently resuspend the pellet in 500 µl SM on a shaker table.
Transfer the phage suspension to a 1.5 ml Epindorf tube.

Add 500µl chloroform to the Epindorf tube. Mix gently.
Spin for 2 minutes at full speed in a microfuge.
Transfer the aqueous (top) phase to a new Epindorf tube. Be careful to avoid the interphase which should be large and well packed.
Add
- 20µl 0.5 M EDTA (pH = 8)
- 5µl 20% SDS
- 20µl 2.5 mg/ml Proteinase K
Incubate at 65° for 30 minutes.
Extract with 1 volume phenol. Centrifuge for 5 minutes and transfer the aqueous phase to a new Epindorf tube.
Extract with 1 volume chloroform:isoamylalcohol (24:1). Centrifuge for 5 minutes and transfer the aqueous phase to a new Epindorf tube.
Add 170µl 6M Ammonium Acetate. Precipitate the DNA with 700µl isopropyl alcohol.
Centrifuge for 15 minutes at 4°.
Rinse twice with 70% ethanol.
Dry the pellet and resuspend in 300µl TE.

For restriction analysis, use 5µl in a 20µl reaction.
Adding RNaseA to a restriction reaction is recommended as there will be a certain amount of RNA mixed in with the phage DNA.
Immediately prior to loading the gel, heat samples to 65° to denature the cos sites.
Reference: Chisolm, D. 1989. A Convenient Moderate-Scale Procedure for Obtaining DNA from Bacteriophage Lambda. BioTechniques 7:21-23.

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