DNA Preparation - Bacteriophage M13 DNA

Jeff Lawrence

1. Inoculate 5 mL 2xYT with 100 mL of a fresh overnight culture of JM101 and a freshly picked plaque. Grow 6 hr at 37°.

2. Pellet cells at 12 K for 12 min in SS34 rotor. Transfer supernatant to a new tube. If cloudy, repellet. Add 1.25 mL of:

	20 %	PEG		(50 mL	40 % PEG)
	2.5 M	NaCl		(50 mL	5 M NaCl)

3. Incubate at room temperature for 30 min. Centrifuge for 10 min at 12 K to pellet phage. Completely remove supernatant. Resuspend pellet in 300 µl PEB:

	300 mM	NaCl		(6 mL	5 N NaCl)
	100 mM	Tris, pH 8.0	(10 mL	1 M Tris, pH 8.0)
	1 mM	EDTA		(200 mL	500 mM EDTA, pH 8.0)
				(84 mL	ddH₂O)

7. Add 300 µl Buffered phenol. Mix well by vortexing. Microfuge for 5 min at 4°. Transfer supernatant to a new tube.

8. Add 300 µl Chloroform (24:1 with Isoamyl alcohol). Mix well by shaking. Microfuge for 2 min. Transfer supernatant to a new tube.

9. Add carrier and mix well. Add 600 µl 95% Ethanol. Precipitate DNA for 2 hr at -20°. 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 50 µl TE. This will yield 1-2 mg/ml. Use 2-3 mL for DNA sequencing.

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

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Eric Kofoid eckofoid at ucdavis.edu