- Gel electrophoresis: Run total of 0.5 µl of the unpurified and 1 µl of the purified dsRNA in 10 µl total volume on a 1% agarose gel. Run 2 µl low mass ladder on a side. Confirm yield and size of single band product.
- Absorbance measurement at 260 and 280 nm: Concentration can be measured using absorbance at 260 nm because it is well established that a solution of RNA with an optical density of 1.0 (1.0 Absorbance Unit) has a concentration of 50 µg/ml in a 10 mm pathlength cell*. A wavelength of 320 nm is used to compensate for the effects of background absorbance due to, for example, turbidity or high absorbance buffer solution.
Concentration = (A260/A320) x 50 x dilution factor, µg/ml
Absorbance ratio can be used to establish the presence of impurities in a sample preparation, relative to a pure sample. The two wavelength of interest to the Molecular Biologist are the absorbance maxima of the nucleic acid, 260 nm, and the protein impurity, 280 nm.
Absorbance ratio = (A260-A320)/ (A280-A320)
The absorbance ratio 1.7 is known for pure nucleotide, enabling rapid assessment of quality. An absorbance ratio of the two wavelengths below the expected 1.7 for the pure substance indicates the presence of impurity in the sample.
To determine concentration and purity of dsRNA, follow simple procedure:
- Fill microvolume cell with water. Set absorbance at 320 nm to zero. This is your background reading.
- Add 2 µl dsRNA to 78 µl water in microvolume cell. Mix by pipetting.
- Measure absorbance at 260, 280 and 320 nm.
- Use formulas for the concentration and for the absorbance ratio to determine concentration and purity of dsRNA.
*Reference: Molecular Cloning, Maniatis et al.