DNA Preparation, Plasmid DNA - CsCl Method


Jeff Lawrence

1. Inoculate 500 mL of fresh media with 3 mL culture and grow overnight at 37°.

2. Centrifuge cells at 8 krpm for 10 min in GSA rotor. Discard supernatant. Resuspend pellet in 5 mL of ice cold:
	25%	Sucrose	50 	(mL	50% Sucrose)
	50 mM	Tris, pH 8.0	(5 mL	1 M Tris, pH 8.0)
				(45 mL	ddH2O)
3. Add 1 µL of 20 mg/mL Lysozyme. Incubate on ice for 10 min. Add 2.8 µL 250 mM EDTA, pH 8.0 and incubate on ice an additional 5 min. Add 8.8 µL of:
	10 %	Triton X-100	(10 mL	Triton X-100)
	50 mM 	Tris, pH 8.0	(5 mL	1 M Tris, pH 8.0)
 	60 mM	EDTA, pH 8.0	(12 mL	500 mM EDTA, pH 8.0)
				(73 mL	ddH2O)
4. Mix well and incubate at 42° until lysis occurs (5-60 min). Centrifuge in T-865 fixed angle rotor at 25 krpm for 90 mi at 5°. Transfer supernatant to a fresh tube. Add 6 µL of:
	30%	PEG		(30 g	PEG)
	1.5 M	NaCl		(30 mL	5 M NaCl)
				(ddH2O to 100 mL)
5. Incubate on ice for 15 min. Centrifuge at 8 krpm for 15 min at 5°. Discard supernatant. Resuspend pellet in 25 µL of 10XTE:
	100 mM	Tris, pH 8.0	(10 mL	1 M Tris, pH 8.0)
	10 mM	EDTA		(2 mL	500 mM EDTA, pH 8.0)
				(88 mL	ddH2O)
6. Add 27 g CsCl to 25 g of the plasmid solution from step 5. Add 1.7 mL 2 mg/mL ethidium bromide. Add solution to a large vTi50 rotor QuickSeal tube. Top off with mineral oil, balance, and seal.

7. Centrifuge to equilibrium at 45 krpm for 18 hr at 15°. Stop rotor with brakes off.

8. Illuminate tube with longwave UV and remove lower band with an 18 guage needle.

9. Remove ethidium bromide with 3-5 extractions with isopropanol saturated with 20xSSC. Repeat until aqueous phase is no longer pink.

10. Add 2.5 volumes of sterile water and 1/20 total volume 4 M NaCl. Add an equal volume of isopropanol and precipitate at -20° for 2 hr.

11. Centrifuge at 10 krpm for 60 min. Discard supernatant. Wash pellet with 70% ethanol. Discard supernatant and air dry. Resuspend pellet in 1 mL TE.


Last Update: Thursday, 19-Jun-2014 11:38:49 PDT
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Eric Kofoid eckofoid at ucdavis.edu