Chloroplast DNA purification using CsCl gradient
Alfred E. Szmidt and Xiao-Ru Wang
- Make up lysate to exactly 4 ml with PTE - buffer and add 4 g CsCl,
dissolve by gentle rolling.
- Add 400 µl of EtBr (ethidium bromide) stock solution (5 mg/ml ),
- 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
- Transfer lysate to Quick-seal Beckman® tubes with Pasteur
- 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
- Centrifuge for 10-16 hrs at 53000 rpm, using VTi 65 - vertical rotor,
- 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 !
- Transfer collected DNA into a plastic tube with stopper.
- 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.
- 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.
- Transfer DNA (together with precipitated CsCl, if any) to a dialysis
tube and close it with clips.
- Dialyze at least 3 times against 4 l of cold 1x TE-buffer, in a cold
room with very gentle stirring.
- Transfer DNA to 1.5 ml Eppendorf tubes and store at -20 C until use.
by Alfred E. Szmidt