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Chapter 14: Nucleic Acids

Exercise 14.10 - Sucrose Density Fractionation

LEVEL II


Figure 14.2 Sucrose gradient distribution of RNA

Materials

Procedure

  1. Refer to Chapter Three for details of fractionation, and form a 10-40% linear sucrose gradient in a nitrocellulose tube.

  2. Dissolve the RNA in 0.02 M sodium acetate solution to yield a final concentration of 250 µg/ml. The low pH of the solution helps to inhibit RNAse, while the salts will keep the RNA from forming large polymeric aggregates. 10

  3. Carefully layer 2.0 ml of the dissolved RNA onto the top of a sucrose gradient. This should be done by slowly allowing the solution to run down the side of the tube and onto the gradient. Be careful not to disturb the gradient.

  4. Load the ultracentrifuge with the prepared tubes and centrifuge for the equivalent of 18,700 RPM for a Beckman SW27 rotor, for 20 hours at 4° C (Refer to Appendix F).

  5. At the completion of centrifugation, remove the tubes and fractionate the contents into 1.0 ml fractions.

  6. Using a UV spectrophotometer 11 and microcuvettes, read the A_2_6_0 of each fraction. Calculate the amount of RNA in each fraction.

  7. Plot the concentration of RNA in each fraction against the fraction number. Based on the density of sucrose in each fraction, compute the density of the RNA in that fraction. Based on the relative size (greater density) of the RNA, determine the nature of the fractions (i.e. rRNA, tRNA).

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu