Exercise 14.9 - Orcinol Determination of RNA

LEVEL I

Materials

• RNA
  
• Alkaline distilled water
  
• Acid-orcinol reagent
  
• Boiling water bath
  
• Spectrophotometer and cuvettes

Procedure

1. Prepare a series of serially diluted RNA standards as in Exercise 14.8, but having a range from 1.0 mg/ml down to 0.125 mg/ml.

2. Prepare a serial dilution of your sample RNA as in Exercise 14.8.

3. Place 3.0 ml of each standard and 3.0 ml of each serial dilution of the sample RNA into separate test tubes. Place 3.0 ml of alkaline water in a separate tube.

4. Add 3.0 ml of acid-orcinol reagent to each tube and mix well.

5. Add 0.3 ml of alcohol-orcinol reagent to each tube and mix well.

6. Place the tubes in a boiling water bath for 20 minutes with marbles placed on top of each tube to prevent evaporation. Cool the tubes by immersion in an ice bath at the end of the 20 minute period.

7. Turn on a spectrophotometer and adjust the wavelength to 660 nm. Blank the spectrophotometer with the alkaline water/orcinol tube. Measure the A of each of the remaining standards and diluted samples.

8. Plot the absorbance of the standards against the known concentrations. Calculate the extinction coefficient, and calculate the concentration of RNA in your sample. Use the dilution yielding an absorbance between 0.1 and 1.5 absorbance units.

Return to Table of Contents