DNA Preparation, Plasmid DNA - SDS Method


Jeff Lawrence

1. Grow 5 mL culture overnight in appropriate antibiotic.

2. Microfuge 1.5 mL of culture for 3 min. Discard supernatant and resuspend pellet in 1 mL TE. Microfuge for 3 min, discard supernatant, and resuspend pellet in 150 µL 1xTAE.

3. Add 300 µL lysing solution:
	3 %	SDS		(30 mL	10 % SDS)
	50 mM	Tris, pH 12.6	(5 mL	1 M Tris, pH 9.0)
				(60 mL	ddH2O)
				(Adjust to pH 12.6 with NaOH)
4. Incubate at 68° for 20-40 min. Add 225 µL 3 M NaOAc, pH 5.3:
	40.8 g 	NaOAc*3H2O
	50 mL	ddH2O
		Glacial Acetic Acid to pH 5.3
		ddH2O to 100 mL final volume
5. Incubate on ice for 60 min. Microfuge for 20 min at 4°. Transfer supernatant to a new tube.

6. Add 500 µL Buffered phenol. Mix well by vortexing. Microfuge for 5 min at 4°. Transfer supernatant to a new tube.

7. Add 500 µL Chloroform (24:1 with Isoamyl alcohol). Mix well by shaking. Microfuge for 2 min. Transfer supernatant to a new tube.

8. Add carrier and mix well. Add 2.5 volumes 95% Ethanol. Precipitate DNA for 1 hr at -20°. Microfuge for 15 min at 4°. Discard supernatant.

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

10. Dry pellet for 4 min in vacuum desiccator. Resuspend pellet in 50 µL TE.


Last Update: Thursday, 19-Jun-2014 11:39:35 PDT
This page has been viewed 102 times.
Eric Kofoid eckofoid at ucdavis.edu