Moderate Scale Lambda Phage DNA Prep

Schedule:

• Prepare a high-titer phage stock
• Isolate phage
• Purify DNA

High-Titer Phage Stock:

1. 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.
2. Incubate at 37° C for 30 minutes
3. Mix phage with 3 ml of NZY Top Agarose (melted and cooled to 50°). Pour onto a small NZY plate.
4. Incubate at 37° overnight.
5. 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.
6. Transfer 1 ml of eluate to a 1.5 ml Epindorf tube.
7. Add 50 µl of CHCl₃ and vortex briefly.
8. Spin in a microfuge for 10 minutes and transfer the aqueous fraction to a new 1.5 ml Epindorf tube.
9. Add 30 µl of CHCl₃. The titer of the phage should be between 10¹⁰ and 5 x 10¹⁰ pfu per ml.

Isolate Phage:

1. Mix 10⁶ phage particles with 500 µl of bacteria (A₆₀₀ = 1 in 10 mM MgSO₄). Incubate at 37° for 30 minutes.
2. Add the preadsorbed phage to 37 ml of NZY in a 125 ml Erlenmeyer flask.
3. Incubate at 37° with vigorous shaking for approximately 5 hours.
4. 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.
5. 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.
6. Add 2.1 g of NaCl and mix gently to dissolve. Place the tube on ice for 5 minutes.
7. Centrifuge at 4° for 20 minutes at 7000 rpm in the Sorval using the SS-34 rotor.
8. Pipet the supernatant to a new Oakridge tube that contains 3.7 g of PEG 8000.
9. Dissolve the PEG with gentle shaking at 37° to 45°.
10. Incubate on ice overnight. You can shorten this step to as little as 1 hour, if necessary.
11. Centrifuge at 4° for 25 minutes at 7000 rpm in the Sorval using the HS-4 rotor.
12. Decant the supernatant and drain the pellet well.
13. Gently resuspend the pellet in 500 µl SM on a shaker table.
14. Transfer the phage suspension to a 1.5 ml Epindorf tube.

Purify DNA:

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

Notes

• 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.

Solutions

SM

Stock Solution	For 500 ml
5 M NaCl	10 ml
1 M Tris-HCl (pH = 7.6)	25 ml
1 M MgS04	5 ml
gelatin	0.05 g

Autoclave

TMM

Stock	For 1 litre
NaCl	5 g
tryptone	10 g
20% maltose	10 ml
1 M MgS04	10 ml

Autoclave

NZY

Stock	For 1 litre
NaCl	5 g
MgSO4·7H2O	2 g
NZ Amine (casein hydrolysate)	10 g
Adjust pH to 7.5

• For Top Agarose add 0.7% (w:v) agarose.
• For plates add 15 g agar.

Autoclave