Cesium Chloride Preparation of DNA

Revision as of 16:14, 8 March 2010 by Davebridges (Talk | contribs) (added concentration to Acetate Buffer)

Revision as of 16:14, 8 March 2010 by Davebridges (Talk | contribs) (added concentration to Acetate Buffer)

Materials

  • GTE Buffer (50 mM Glucose, 25 mM Tris, 10 mM EDTA)
  • Lysis Solution (0.2N NaOH, 1% SDS)
  • Sodium Acetate (3M pH 4.8 and 3M pH 7; pH with Acetic Acid)
  • 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