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        Spectrophotometric Analysis of rRNA

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        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.

         

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