Chloroplast DNA purification using CsCl gradient

Alfred E. Szmidt and Xiao-Ru Wang

  1. Make up lysate to exactly 4 ml with PTE - buffer and add 4 g CsCl, dissolve by gentle rolling.
  2. Add 400 µl of EtBr (ethidium bromide) stock solution (5 mg/ml ), mix gently.
  3. If you have refractometer at hand then bring samples to refractive index: 1.3880-1.3900 with PTE or CsCl; otherwise skip this step. We skip it always.
  4. Transfer lysate to Quick-seal Beckman® tubes with Pasteur pipette.
  5. Switch on tube sealing device. Balance and seal tubes. Be careful here, the tubes weight should not differ by more than 0.001 g. Check if all tubes are properly sealed, by wrapping in Kleenex® and squeezing hardly in hand.
  6. Centrifuge for 10-16 hrs at 53000 rpm, using VTi 65 - vertical rotor, (Beckman® ultracentrifuge).
  7. Clip off the tube top and collect DNA band with 1ml plastic syringe (large bore needle). To improve visibility of DNA band illuminate tubes with UV while performing this step; wear glasses !
  8. Transfer collected DNA into a plastic tube with stopper.
  9. To remove EtBr extract by adding equal volume of isopropanol saturated with CsCl followed by gentle rolling. Discard upper pink phase. Sometimes, CsCl may precipitate during this step. Do not bother with it as CsCl will be later removed by dialysis.
  10. Repeat p. 10 until the pink color in the upper phase is gone. With very high DNA yields it may be difficult to completely remove EtBr. If that happens, proceed to p. 12 anyway. We found that small amounts of EtBr do not affect our DNA extracts.
  11. Transfer DNA (together with precipitated CsCl, if any) to a dialysis tube and close it with clips.
  12. Dialyze at least 3 times against 4 l of cold 1x TE-buffer, in a cold room with very gentle stirring.
  13. Transfer DNA to 1.5 ml Eppendorf tubes and store at -20 C until use.

by Alfred E. Szmidt