DNA Preparation - Bacteriophage Lambda DNA

Jeff Lawrence

1. Prepare phage stock as described in 'Purification of Phage Lambda.'

2. To the 600 µl phage stock add 10 µl 0.1 mg/ml Proteinase K and 24 µl 10% SDS. Mix and incubate at room temperature for 10 minutes.

3. Add 100 µl 3 M KOAc, pH 8.5. Heat to 88° for 20 min. Cool to room temperature.

4. Incubate on ice for 15 min. Centrifuge at 12 krpm (12 kg) for 15 min at 4°.

5. Transfer supernatant to a fresh tube.

6. Add 700 µl Buffered phenol. Mix well by vortexing. Microfuge for 5 min at 4°. Transfer supernatant to a new tube.

7. Add 700 µl Chloroform (24:1 with Isoamyl alcohol). Mix well by shaking. Microfuge for 2 min. Transfer supernatant to a new tube.

8. Add carrier and mix well. Add 700 µl Isopropanol. Precipitate DNA at -20° for 2 hr. Microfuge for 15 min at 4°. Discard supernatant.

10. Add 1 mL 70% Ethanol. Rinse pellet well, dislodging it from the bottom of the tube. Microfuge for 2 min. Discard supernatant.

11. Dry pellet for 4 min in vacuum desiccator. Resuspend pellet in 40 µl TE.

Last Update: Thursday, 12-Dec-2024 00:55:19 UTC

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Eric Kofoid eckofoid at ucdavis.edu