CsCl-Density Separation of DNA

• DNA
• CsCl
• 0.3 N NaOH
• 0.2 M Tris-HCl buffer, pH 7.0
• Ultracentrifuge and rotor
• UV spectrophotometer and cuvettes

1. Determine the G+C content of the sample DNA.
2. Once the G+C content is determined, the buoyant density of the DNA can be determined from the formula:
   
   p = 1.660 g/cm³ + 0.098 × (G+C fraction)
   Determine the concentration of CsCl salts to use for dissolution of the DNA.
3. Dissolve approximately 100 micrograms of DNA in 4.2 mL of the appropriate CsCl solution in 0.3 N NaOH.
4. Load the dissolved DNA/CsCl solution onto a centrifuge tube suitable for a 4.2 mL sample and speeds of 30–40000 rpm (Beckman SW39 rotor, or equivalent).
5. For the Beckman SW39 rotor, centrifuge the material at 35000 rpm for 65 hours at 22°C.
6. Collect the fractions in 0.1-mL steps.
7. Add 0.2 M Tris-HCl, pH 7.0 to each fraction and measure the A₂₆₀ for each fraction. If available, a continuous flow system using a fraction collecting device may be used.