Purification of Closed Circular DNA by Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradients: Discontinuous 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 30C 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 μg/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 20C. 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 1 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 1 band. Allow viscous DNA to flow out into a tube. Plug the needle with modeling clay. 13. Leaving the 2 needle in place, insert a 3 hypodermic needle (18-gauge) into the lower band and collect into a tube. 14. Proceed with removal of Ethidium bromide from purified DNA