DNA Preparation, Plasmid DNA - Alkaline Method


Jeff Lawrence

1. Grow 5 mL culture overnight in appropriate antibiotic.

2. Microfuge 1.5 mL of culture for 3 min. Remove supernatant by aspiration and resuspend pellet in 200 µL of NET:
	100 mM	NaCl		(2 mL	5 N NaCl)
	10 mM	Tris, pH 8.0	(1 mL	1 M Tris, pH 8.0)
	10 mM	EDTA		(2 mL	500 mM EDTA, pH 8.0)
				(95 mL	ddH2O)
3. Microfuge for 3 min. Discard supernatant. Resuspend pellet in 100 µL of ice cold GTE:
	50 mM	Glucose		(2 mL	50% Glucose)
	25 mM	Tris, pH 8.0	(2.5 mL	1 M Tris, pH 8.0)
	10 mM	EDTA		(2 mL	500 mM EDTA, pH 8.0)
				(93.5 mL	ddH2O)
4. Add 200 µL of:
	200 mM	NaOH		(4 mL	5 N NaOH)
	1 % 	SDS		(5 mL	20% SDS)
				(91 mL	ddH2O)
5. Incubate on ice for 5 min. Add 150 µL ice cold 3 M KOAc:
	60 mL	5 M KOAc
	11.5 mL	Glacial acetic acid
	28.5 mL	ddH2O
6. Incubate on ice for 5 min. Microfuge for 15 in at 4°. Transfer supernatant to a new tube.

7. Add 450 µL Buffered phenol. Mix well by vortexing. Microfuge for 5 min at 4°. Transfer supernatant to a new tube.

8. Add 450 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 1 mL 95% Ethanol. Incubate at room temperature for 3 min. 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.


Last Update: Thursday, 19-Jun-2014 11:38:27 PDT
This page has been viewed 227 times.
Eric Kofoid eckofoid at ucdavis.edu