CsCl/EtBr Density Gradient Centrifugation

CsCl/EtBr Density Gradient Centrifugation

Caution: Wear gloves because of EtBr and CsCl. EtBr is strong mutagenic and contact with it should be avoided. CsCl is a heavy metal and its exposure should be limited. In case of spill, rinse the contaminated area with copious amount of H2O.
  1. The same as described in Cotton DNA extraction
  2. Add one volume of ice cold isopropanol (usu. up to 40 ml), invert or rock the tubes gently and sit tubes for at least 15 min. You should see the DNA strands or clump in the tubes.
  3. Spin the tubes (3K, 20 min).
  4. Drain the supernatent. Becareful not to lose the pellets. Pellets may be clear and colorless.
  5. Rinse the pellets with 70 % EtOH and drain for 5 min at room temp.
  6. Dry the pellets under vacuum for 5 min.
  7. Resuspend the pellets in 10 ml TE very well. You may use DPTP to break the pellets to suspend in the buffer.
  8. Take 8.9 ml DNA suspension into a 10 ml cylinder.
  9. Add 8.3 g CsCl to each 8.9 ml, dissolve, add 0.3 ml 10 mg/ml EtBr, and dissolve completely. CsCl may not go into the DNA suspension for a while. But it goes into the buffer: Be patient.
  10. Fill a 70.1 Ti polyallomer centrifuge tube using a cut-off cannula and top off the tube with TE using a pasteur pipet. In order to avoid the forming bubbles in the tube, put the cannula below the sealing ring at the bottom of the neck, cock the syringe to the side of the neck to allow air to escape, and pour the all the liquid in at once.
  11. For pouring the next sample in the other tube, have the outside of the cannula and the neck of the centrifuge tube dry to avoid the solution from bubbling out.
  12. Weigh the tubes and balance them: if they are not between 26.8 g to 27.9 g, something is probably wrong, check.
  13. Spin at 53 K RPM for at least 18 hr.
  14. Remove the DNA band(s) with a 19 gauge needle in minimal volume, usually about 1 ml. Plasmid(supercoiled DNA) band is the bottom one in a CsCl/EtBr gradient. Remove the top to the tube before sucking out the sample. Save the CsCl.