Exercise 14.6 - CsCl - Density Separation of DNA
- 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 (Exercise 14.5)
- Once the G+C content is determined, the bouyant density of
the DNA can be determined from the formula:
p = 1.660 g/cm
+ 0.098 x (G+C fraction)
Using the bouyant density and the CsCl Table in Appendix F,
determine the concentration of CsCl salts to use for dissolution
of the DNA.
- 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 30-40,000 RPM (Beckman
SW39 rotor, or equivalent).
- For the Beckman SW39 rotor, centrifuge the material at
35,000 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
for each fraction. If available, a continuous flow system using a
fraction collecting device may be used.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org