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 June 19 2014
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Eric Kofoid
eckofoid at ucdavis.edu