Purification of Plasmid and Genomic DNA

Purification of Closed Circular DNA by Equilibrium Centrifugation in

CsCl-Ethidium Bromide Gradients

A. Continuous Gradients
1. Measure the volume of the DNA solution. For every milliliter, add exactly 1 g of
solid CsCl.
2. Warm solution to 30oC to facilitate the dissolution of the salt. Mix until
dissolved
3. Add 0.8 mL of ethidium bromide (10 mg/mL in water) for every 10 mL of
DNA/CsCl solution.
4. Mix the ethidium bromide solution with the denser DNA/CsCl solution.
5. The final density should be 1.55 g/mL and the concentration should of ethidium
bromide should be 740 g/mL
6. Centrifuge the solution at RT at 8000 rpm in a sorvall SS34 rotor (or equivalent)
7. Remove the furry scum which floats to the surfacte with a large-gauge needle.
This consists of complexes formed b/w ethidium bromide and bacterial proteins
8. Transfer red solution to a tube suitable for ultracentrifugation
9. Fill remainder of the tube with light paraffin oil and seal the tube
10. Centrifuge the density gradients at 45,000 rpm for 16 hours (VTi65), 45,000 rpm
for 48 hours (Ti50), 60,000 rpm for 24 hours (Ti65) or 60,000 rpm for 24 hours
(Ti70.1) at 20oC.
11. Two bands of DNA, located in the center of the gradient, should be visable. The
upper band is nicked circular DNA and linear bacterial DNA. The lower, larger
band consists of closed circular plasmid DNA.
12. Pierce top of tube with 21-gauge needle to break vacuum. Collect upper band 1st
to prevent contamination by placing a strip of scotch tape on the side of the tube
and then inserting an 18-gauge needle through the tape into the 1st band. Allow
viscous DNA to flow out into a tube. Plug the needle with modeling clay.
13. Leaving the 2nd needle in place, insert a 3rd hypodermic needle (18-gauge) into the
lower band and collect into a tube.
14. Proceed with removal of Ethidium bromide from purified DNA

B. Discontinuious Gradient
1. Prepare a solution of CsCl ( = 1.47 g/mL) by adding 125 g of CsCl to 167 mL of
TE
2. Add 8 mL of CsCl solution to ultracentifuge tube and set aside.
3. Adjust volume of DNA solution to 3 mL with TE (pH 8.0)
4. Add 8.4 g of CsCl to the solution of DNA and warm to 30oC to facilitate
dissolution of salt.
5. Weigh the solution and add TE (pH 8.0) until the solution wights exactly 13.2 g
6. Add 0.8 mL of Ethidium bromide (10 mg/mL in Water) and quickly mix
7. Centrifuge at 8000 rpm for 5 minutes in Sorvall SS34 Rotor (or equivalent).
8. Remove furry scum from top of solution
9. Place a 9 Pasteur pipette into the centrifuge tube containing CsCl from step B2
such that the tip is touching the bottom of the tube. Carefully load the clear, red
 Purification of Plasmid and Genomic DNA

solution from step B8 through the Pasteur pippet so that the sample is layered
underneath the 1.47 g/mL CsCl solution.
10. If necessary, fill the tube with CsCl solution prepared in step B1.
11. Load sample into Beckman Ti70.1 or Sorvall 65.13 rotor (0r equivalent) and
centrifuge at 60,000 rpm for 6 hrs at 20oC
12. Recover band as described in A12-13
13. Proceed with removal of Ethidium bromide form purified DNA