The most important contaminants in λ DNA prepared from plate lysates are polyanions (i.e., bacterial nucleic acids and restriction endonuclease inhibitors from agar). Conventional methods for purifying λ DNA are not entirely successful at removing these contaminants. These methods are frequently time-consuming and have the additional disadvantages of requiring either nuclease treatment, phenol-chloroform extraction or high-speed centrifugation.
Since the λ DNA is initially encapsulated in viral coat proteins, it does not bind to anion exchange resins. This allows the contaminants mentioned above to be removed by absorption on DE52 cellulose. Once the DE52 cellulose has been removed, the λ DNA can be isolated using a modified protocol for the Wizard® DNA Clean-Up System.
Phage Purification Using Whatman® DE52
SM buffer: 50mM Tris-HCl (pH7.5), 100mM NaCl, 8mM MgSO4
- Pick a single plaque into 1ml SM buffer and incubate overnight at 4°C. Use 0.1ml of this phage suspension (approximately 105–106pfu) to infect 0.3ml of 0.6 OD600 bacterial suspension or use 0.1ml from a 1:1,000 dilution of a plate lysate.
- Incubate at 37°C for 30 minutes. Plate onto a 140mm plate in 0.7% top agarose. When the plate is set, incubate at 37°C until confluent lysis has occurred (approximately 6 hours).
- Add 10ml of 0.01% gelatin in SM buffer. Incubate overnight at 4°C.
- Collect the supernatant, and wash the plate surface with SM buffer to bring the volume to 10ml. Add 0.1ml of CHCl3, and mix by inversion.
- Centrifuge at 3,000rpm for 30 minutes at 4°C.
- Collect the supernatant in a 15ml centrifuge tube. Add 50µl of 0.1% bromophenol blue and 5ml of a 50% suspension of Whatman® DE52 equilibrated in SM buffer. Mix occasionally by inversion until the dye, acting as an indicator for charged material, also binds to the DE52 (in approximately 5–10 minutes). Centrifuge at 3,000rpm for 5 minutes at room temperature. Proceed with DNA purification.
Lambda DNA purification Using the Wizard® DNA Clean-Up System
This procedure is a modification of the protocol from the Wizard® DNA Clean-Up System Technical Bulletin (#TB141) for DNA purification without a vacuum manifold.
- Place 0.8g guanidine thiocyanate per ml of supernatant (10g/12.5ml) into a 30ml centrifuge tube. Add the supernatant from step 6 of the phage purification protocol.
- Warm the Wizard® DNA Clean-Up Resin at 37°C for 10 minutes. Add 1ml of fully resuspended resin to the guanidine thiocyanate, and mix gently by inverting the tube until the guanidine thiocyanate is fully dissolved (at least 15 minutes).
- Concentrate the resin, and remove microsomes by accelerating in a centrifuge to 1,000rpm and switching off. Remove the supernatant down to approximately 5ml.
- Gently resuspend the resin in the residual supernatant. Remove the plunger from a disposable syringe, and attach a Wizard® Minicolumn to the syringe barrel. Pipet the Wizard® λ DNA Clean-Up Resin containing the bound DNA into the syringe barrel. Insert the syringe plunger slowly, and gently push the slurry into the Minicolumn.
- Detach the syringe from the Minicolumn, and remove the plunger from the syringe. Reattach the syringe barrel to the Minicolumn. To wash the column, pipet 2ml of 80% isopropanol into the syringe. Insert the plunger into the syringe, and gently push the solution through the Minicolumn.
- Remove the syringe barrel, and transfer the Minicolumn to a 1.5ml microcentrifuge tube. Centrifuge the Minicolumn for 2 minutes at maximum speed (10,000 x g) to dry the resin.
- Transfer the Minicolumn to a new microcentrifuge tube. Apply 100µl of distilled water prewarmed to 80°C to the Minicolumn, and wait 1 minute. Centrifuge the Minicolumn for 20 seconds at maximum speed (10,000 x g) to elute the λ DNA. For maximum recovery repeat the elution step and combine eluates. This protocol yields approximately 12µg of λ DNA.
Results and Conclusions
This procedure is simple, reliable and rapid. Although the use of bromophenol blue to monitor absorption on the ion exchanger is not essential, it provides a reassuring check for this step. Finally, care should be taken to avoid transferring any of the DE52 into the subsequent steps, as this would probably reduce yields of λ DNA.
Thank you to Dr. Winterbourne at the Department of Surgery, St. Georges Medical School, London for this protocol.