Exercise 14.8 - Spectrophotometric Analysis of rRNA

LEVEL I

Materials

• RNA
  
• UV spectrophotometer and cuvettes
  
• Alkaline distilled water

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 of each of the standards. Plot the A 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.