Method

1. Use a borosilicate Pasteur pipette to pick a single well-isolated bacteriophage plaque into 1 ml of SM containing a drop of chloroform in a small sterile polypropylene tube. Store the suspension for 4-6 hours at 4°C to allow the bacteriophage particles to diffuse from the top agarose.

2. In a 25-ml tube, mix 0.5 ml of the bacteriophage suspension (approx. 3 x 106 bacteriophages) with 0.1 ml of an overnight culture of bacteria. Incubate the culture for 15 minutes at 37°C.

3. Add 4 ml of NZCYM medium, and incubate the culture for approx. 9 hours at 37°C with vigorous agitation.

The culture should be clear, but very little debris should be evident.

4. Add 0.1 ml of chloroform to the culture and continue incubation for a further 15 minutes at 37°C with vigorous agitation. Transfer the lysate to a 5-ml polypropylene centrifuge tube. Centrifuge at 800g (2600 rpm in a Sorvall SS-34 rotor) for 10 minutes at 4°C.

5. Transfer the supernatant to a fresh tube, and remove the bacterial debris by centrifugation at 4000g (5800 rpm in a Sorvall SS-34 rotor) for 10 minutes at 4°C. A small aliquot of cleared lysate can be set aside at this step as a bacteriophage stock solution. Store the stock at 4°C over chloroform.

6. Dispense 10 ml of a 2:1 slurry of DE52 resin into a fresh centrifuge tube and sediment the resin by centrifugation at 500g (2000 rpm in a Sorvall SS-34 rotor) for 5 minutes at room temperature. Remove the supernatant from the resin pellet and place the centrifuge tube on ice.

7. Resuspend the DE52 in the cleared bacteriophage A supernatant and allow the bacteriophage particles to absorb to the resin by rocking the centrifuge tube for 3 minutes at room temperature.

8. Centrifuge the bacteriophage A supernatant/DE52 slurry at 4000g (5800 rpm in a Sorvall SS-34 rotor) for 5 minutes. Carefully transfer the supernatant to a fresh centrifuge tube and repeat the centrifugation step. Discard the pellet after each centrifugation.

9. Transfer the supernatant from the second centrifugation to a fresh centrifuge tube. Extract the supernatant, which contains the bacteriophage A particles, once with phenol:chloroform.

10. Transfer the aqueous phase, which contains the bacteriophage A DNA, to a fresh polypropylene tube and add an equal volume of isopropanol. Store the mixture for 10 minutes at -70°C.

11. Collect the precipitated bacteriophage DNA by centrifugation at 16,500g (12,000 rpm in a Sorvall SS-34 rotor) for 20 minutes at 4°C.

12. Drain the isopropanol from the centrifuge tube and allow the pellet of DNA to dry in the air.

13. Dissolve the DNA pellet in 2 ml of low-salt buffer.

14. Purify the bacteriophage DNA by chromatography on an Elutip-d column as described in Chapter 2, Protocol 23 .

15. Mix 1 ml of ethanol with the solution of eluted DNA and incubate the mixture on ice for 20 minutes. Collect the precipitated DNA by centrifugation in a microfuge, discard the supernatant, and rinse the pellet of DNA with 0.5 ml of 70% ethanol. Discard the supernatant and allow the ethanol to evaporate. Dissolve the damp pellet of DNA in 50 pl of TE (pH 8.0).

Resuspend the DNA by tapping on the side of the tube. Try to avoid vortexing. If the DNA proves difficult to dissolve, incubate the tube for 15 minutes at 50°C.

If minipreparations are working well, expect to isolate approx. 5 pg of purified bacteriophage DNA from 5 X 1010 infectious particles. It is possible to estimate the quantity of bacteriophage DNA present in a plate lysate or liquid culture lysate by direct agarose gel electrophoresis (please see Chapter 2, Protocol

For restriction analysis and agarose gel electrophoresis, digest a 5-10-pl aliquot of the resuspended DNA.

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