DNA Preparation - Bacterial DNA


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 µL	500 M 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 30 µl 10% SDS and incubate on ice for an additional 2 min.

5. Place tubes at 65° for 15 min then cool to room temperature.

6. Add 400 µl Buffered phenol. Mix well by vortexing and microfuge for 15 min at 4°. Transfer supernatant to a new tube. Avoid taking any material at the interface.

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.

Begin, Optional Rnase section
10. Dry in vacuum desiccator for 4 min or until just dry. Do not overdry. Resuspend pellet in 200 µL TE. As an option, RNA may be removed as described below.

11. 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°.

12. Add 500 µL 95% ethanol and mix by inversion. Microfuge for 3 minutes. Discard supernatant.
End, Optional Rnase section

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

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


Last Update: Thursday, 19-Jun-2014 11:29:09 PDT
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Eric Kofoid eckofoid at ucdavis.edu