- 0.3 N NaOH
- 0.2 M Tris-HCl buffer, pH 7.0
- Ultracentrifuge and rotor
- UV spectrophotometer and cuvettes
- Determine the G+C content of the sample DNA.
- 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
- Dissolve approximately 100 micrograms of DNA in 4.2 mL of the appropriate
CsCl solution in 0.3 N NaOH.
- 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
- For the Beckman SW39 rotor, centrifuge the material at 35000 rpm for 65
hours at 22°C.
- Collect the fractions in 0.1-mL steps.
- 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.