DNA Preparation, Plasmid DNA - Boiling Method


Jeff Lawrence

1. Grow 3 mL culture overnight in appropriate antibiotic. Pellet cells at 12 K for 10 min in table top centrifuge. Remove supernatant by aspiration. Resuspend pellet in 400 µL STET:
	8 %	Sucrose		(40 mL	20% Sucrose)
	10 mM	Tris, pH 8.0	(1 mL	1 M Tris, pH 8.0)
	50 mM	EDTA		(10 mL	500 mM EDTA, pH 8.0)
	0.5 %	Triton X-100	(5 mL	10% Triton X-100)
				(44 mL	ddH2O)
2. Add 20 µL of 20 mg/mL Lysozyme. Incubate at room temperature for 1 min.

3. Add 2 µL fresh DEPC. Vortex 30 sec.

4. Boil immediately for 40 sec. Chill quickly on ice.

5. Microfuge for 15 min at 4°. Remove pellet with a sterile toothpick or transfer supernatant to a new tube.

7. Add 1 mL 95 % Ethanol. Precipitate for 30 min at -20°. Microfuge for 15 min at 4°.

8. Discard supernatant. Dry in vacuum desiccator for 4 min. Resuspend pellet in 50 µL TE. DNA prepared by this method may not be clean enough for restriction digests.


Last Update: Thursday, 19-Jun-2014 11:38:39 PDT
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Eric Kofoid eckofoid at ucdavis.edu