DNA Preparation, Plasmid DNA-LiCl Method


Jeff Lawrence

1. Grow 5 mL culture overnight in appropriate antibiotic. Inoculate 100-250 mL of fresh media with 3-5 mL culture and grow to Klett 150 : end log growth.

2. Centrifuge cells at 8 krpm for 10 min in GSA rotor; resuspend pellet in 10 mL TE. Centrifuge at 10 krpm for 8 min in SS34 rotor; resuspend pellet in 3 mL of:
	0.23 g	Glucose
	0.30 g 	Lysozyme
	0.5 mL	500 mM EDTA
	0.6 mL	1 M Tris, pH 8.0
	23.9 mL	ddH2O
3. Incubate on ice for 30 min. Add 10 mL DEPC and 6 mL of:
	4 mL	5 N NaOH
	5 mL	10 % SDS
	91 mL	ddH2O
4. Incubate on ice an additional 5 min. Add 4.5 mL 3 M NaOAc, pH 5.8 and incubate on ice an additional 30 min.

5. Add 35 mL 95% Ethanol. Precipitate DNA at -80° for 30 min. Centrifuge at 10 krpm for 15 min. Discard supernatant; air dry. Add 1 mL 8 M LiCl and 3 mL NT:
	2 mL	5 N NaCl
	5 mL	1 M Tris, pH 8.0
	93 mL	ddH2O
6. Incubate on ice for 20 min. Centrifuge at 10 krpm for 10 min and recover the supernatant. Add 9 mL 95% Ethanol. Mix well and precipitate at -20° for 2 hr. Centrifuge at 10 krpm for 15 min. Discard supernatant and air dry.

7. Resuspend pellet in 300 µL TE. Add 10 µL 2 mg/ml RNase (boiled 10 min prior to first use) and incubate at 37° for 30 min.

8. Add 300 µL Buffered phenol. Mix well by vortexing. Microfuge for 5 min at 4°. Transfer supernatant to a new tube.

9. Add 300 µL Chloroform (24:1 with Isoamyl alcohol). Mix well by shaking. Microfuge for 2 min. Transfer supernatant to a new tube.

10. Add carrier and mix well. Add 600 µL 95% Ethanol. Precipitate DNA for 2 hr at -20°. Microfuge for 15 min at 4°. Discard supernatant.

11. Add 1 mL 70% Ethanol. Rinse pellet well, dislodging it from the bottom of the tube. Microfuge for 2 min. Discard supernatant. Air dry pellet and resuspend in 100 µL TE.


Last Update: Thursday, 19-Jun-2014 11:39:09 PDT
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Eric Kofoid eckofoid at ucdavis.edu