Spectrophotometric Analysis of rRNA

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 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.

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