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Chapter 14: Nucleic Acids

Exercise 14.8 - Spectrophotometric Analysis of rRNA

LEVEL I

Materials

Procedure

  1. Dissolve 10 mg of commercial RNA in 250 ml. of slightly alkaline distilled water. Use a volumetric flask and proper analytical technique. This will give a standard solution of 40 micrograms RNA/ml.

  2. Prepare a series of dilutions so that you have 40, 20, 10, 5 and 2.5 micrograms of RNA per ml.

  3. Turn on a UV spectrophotometer and adjust the wavelength to 260 nm. Use the alkaline water to blank the spectrophotometer at 260 nm.

  4. Read the A_2_6_0 of each of the standards. Plot the A_2_6_0 vs the concentration of RNA and calculate the extinction coefficient.

  5. Dissolve your isolated, precipitated RNA in 10.0 ml of alkaline water. Prepare a serial dilution for 1/10, 1/100, 1/1000 and 1/10000. Measure the absorbance of each at 260 nm and, using the dilution which gives a reading between .1 and 1.5 absorbance units, compute the concentration of RNA in your sample.

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