Spectrophotometric Analysis of rRNA

• RNA
• UV spectrophotometer and cuvettes
• Alkaline-distilled water

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 yield 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 A₂₆₀ of each of the standards. Plot the A₂₆₀ versus 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 that produces a reading between .1 and 1.5 absorbance units, compute the concentration of RNA in your sample.