DNA isolation from lysates (Promega)

The Phage Cookbook by Dominik Refardt

DNA isolation from λ lysates with the Wizard^® DNA clean-up system

λ DNA can be purified from plate lysates by an initial purification use DEAE-cellulose (Whatman^® DE52) followed by λ DNA purification using the Wizard DNA Clean-Up System (Promega).

Introduction

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.

Method

Phage Purification Using Whatman^® DE52

SM buffer: 50 mM Tris-HCl (pH7.5), 100 mM NaCl, 8 mM MgSO₄

• 1. Pick a single plaque into 1ml SM buffer and incubate overnight at 4 °C. Use 0.1 mL of this phage suspension (approximately 10⁵–10⁶pfu) to infect 0.3ml of 0.6 OD₆₀₀ bacterial suspension or use 0.1 mL from a 1:1,000 dilution of a plate lysate.

  2. Incubate at 37 °C for 30 minutes. Plate onto a 140 mm plate in 0.7% top agarose. When the plate is set, incubate at 37 °C until confluent lysis has occurred (approximately 6 hours).

  3. Add 10 mL of 0.01% gelatin in SM buffer. Incubate overnight at 4°C.

  4. Collect the supernatant and wash the plate surface with SM buffer to bring the volume to 10 mL. Add 0.1 mL of CHCl₃ and mix by inversion.

  5. Centrifuge at 3,000 rpm for 30 minutes at 4 °C.

  6. Collect the supernatant in a 15-mL centrifuge tube. Add 50 µl of 0.1% bromophenol blue and 5 mL 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 3,000 rpm for 5 minutes at room temperature. Proceed with DNA purification.

λ 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.

• 1. Place 0.8 g guanidine thiocyanate per ml of supernatant (10 g/12.5 mL) into a 30-mL centrifuge tube. Add the supernatant from step 6 of the phage purification protocol.

  2. Warm the Wizard^® DNA Clean-Up Resin at 37 °C for 10 minutes. Add 1 mL 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).

  3. Concentrate the resin and remove microsomes by accelerating in a centrifuge to 1,000 rpm and switching off. Remove the supernatant down to approximately 5 mL.

  4. 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.

  5. 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 2 mL of 80% isopropanol into the syringe. Insert the plunger into the syringe and gently push the solution through the Minicolumn.

  6. Remove the syringe barrel and transfer the Minicolumn to a 1.5-mL microcentrifuge tube. Centrifuge the Minicolumn for 2 minutes at maximum speed (10,000 x g) to dry the resin.

  7. Transfer the Minicolumn to a new microcentrifuge tube. Apply 100 µl 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 12 µg of λ DNA.

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.