Ethanol precipitation efficiently removes unwanted components from the ShortCut digestion and quantitatively yields the siRNA in a small pellet.
- Add one-tenth volume of 3 M NaOAc (pH 5.5), 2 µl RNase-free Glycogen and 3 volumes of cold 95% ethanol. Place at -70°C for 30 minutes or -20°C for 2 hours.
- Spin for 15 minutes in a microcentrifuge at 14,000 rpm.
- Remove supernatant; to the pellet, add two volumes 80% ethanol, incubate at room temperature for 10 minutes, centrifuge for 5 minutes, decant the tube.
- Allow the pellet to air-dry.
- Dissolve the dried RNA in 10 mM Tris-HCl (pH 7.0), 1 mM EDTA, or dH20 to approximately 1/3 volume of the original ShortCut digestion.
The siRNA solution can be quantitated by UV absorbance, comparison on agarose gel to DNA standards or fluorescence.