Cesium Chloride Preparation of DNA

Materials

• GTE Buffer (50 mM Glucose, 25 mM Tris, 10 mM EDTA)
• Lysis Solution (0.2N NaOH, 1% SDS)
• Sodium Acetate (pH 4.8 and 3M pH 7)
• Ultracentrifuge tubes (Beckman 362185)
• Water-Saturated N-butanol
• Absolute ethanol

Protocol

1. Grow 1L overnight culture in 2XYT media with antibiotic
2. Pellet and resuspend in 20 mL of GTE buffer
3. Add 40 mL Lysis Solution for 5 minutes
4. Add 30 mL Sodium Acetate pH 4.8 for 10 minutes
5. Spin in JA-10 for 20 min at 8000 RPM
6. Remove supernatant and add 0.7 vol Isopropanol
7. Spin 20 min at 8000 RPM
8. Resuspend DNA in 6 mL of distilled water. Add 10g of CsCl and dissolve.
9. Load into two ultracentrifuge tubes then add 25 uL EtBR to each tube
10. Centrifuge 60 000 RPM (250 000g) O/N in NVT90 rotor
11. Withdraw the upper band with a 18g needle and reload into 1g/L CsCl for second spin
12. Remove band and extract EtBr with butanol until clear
13. Dialyse overnight in 2L of TE
14. Precipitate with 3 vol absolute ethanol and 1/10 vol of 3M Acetate pH 7
15. Resuspend to a final concentration of 5-10 mg/mL in TE