Moderate Scale Lambda Phage DNA Prep
Based on a protocol I got from Rogene Schnell
- Prepare a high-titer phage stock
- Isolate phage
- Purify DNA
High-Titer Phage Stock:
- 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.
- 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.
|Stock Solution||For 500 ml|
|5 M NaCl||10 ml|
|1 M Tris-HCl (pH = 7.6)||25 ml|
|1 M MgS04||5 ml|
|Stock||For 1 litre|
|20% maltose||10 ml|
|1 M MgS04||10 ml|
|Stock||For 1 litre|
|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.
This page is, as always, Copyright (c) 1997 by Craig D. Amundsen. All rights reserved.