DNA Preparation - Bacterial DNA from Mucoid Strains

Jeff Lawrence

1. Grow 5 mL culture overnight. Pellet 1.5 mL culture in a microcentrifuge tube for 3 min. Remove supernatant with a vacuum aspirator and resuspend pellet in 1 mL TE:

	10 mM	Tris, pH 8.0	(1 mL	1 M Tris, pH 8.0)
	1 mM	EDTA		(200 mL	500 mM EDTA, pH 8.0)
				(99 mL	ddH2O)

2. Microfuge as above, remove supernatant, and resuspend pellet in 400 µL NET:

	100 mM 	NaCl		(2 mL	5 N NaCl)
	50 mM 	EDTA		(10 mL	500 mM EDTA, pH 8.0)
	50 mM	Tris, pH 8.0	(5 mL	1 M Tris, pH 8.0)
				(83 mL	ddH2O)

3. Add 25 µL 20 mg/mL Lysozyme in NET. Incubate on ice for 10 min.

4. Add 15 µL 20% SDS and incubate on ice for an additional 2 min.

5. Add 40 µL CTAB solution. Incubate at 65° for 15 min then cool to room temperature. CTAB is:

 	700 mM	NaCl		(4.1 g	5 N NaCl)
	10 % 	CTAB		(10.0 g	Hexadecyltrimethylammonium bromide)
				(83 mL	ddH2O)

6. Add 400 µL Buffered phenol. Mix well by vortexing and microfuge for 15 min at 4°. Transfer supernatant to a new tube.

7. Add 400 µL Chloroform (24:1 with Isoamyl alcohol). Mix well by shaking and vortexing and microfuge for 2 min. Transfer supernatant to a new tube.

8. Add 850 µL 95% Ethanol and mix by inversion. DNA should precipitate immediately. Microfuge for 3 minutes.

9. Discard supernatant. Dry in vacuum desiccator for 4 min or until just dry. Do not overdry. Resuspend pellet in 200 µL TE.

10. Add 8 µL 5 N NaCl and 5 µL 2 mg/mL RNase (boiled 10 min before first use). Incubate for 30 min at 37°.

11. Add 500 µL 95% ethanol and mix by inversion. Microfuge for 3 minutes. Discard supernatant.

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

13. Dry in vacuum desiccator as above. Resuspend pellet in 100 µL TE. Yield is 0.5-2.0 mg/mL.

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

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Eric Kofoid eckofoid at ucdavis.edu