Protocol for Annealing for dsRNA

1. Resuspend each RNA oligo to a concentration of 50 µM.
2. Combine 30 µl of each RNA oligo solution and 15 µl of 5X Annealing Buffer. Final volume is 75 µl.
3. Incubate the solution for 1 minute at 90° C .
4. Centrifuge the tube for 15 seconds to bring the solution to the bottom.
5. Allow to cool slowly to room temperature. (This is best accomplished using a beaker of water as a waterbath to achieve the 90°C temperature and then removing the heat source and allowing the beaker of water containing the tube of oligos to cool slowly to room temp.)
6. The solution can be stored frozen at 20° C and freeze-thawed up to 5 times. The final concentration of siRNA duplex is 20 µMolar.

5X Annealing Buffer:
50 mM Tris-HCl, pH 8.0
100 mM NaCl
5 mM EDTA

For additional information, click RNA Oligo Supporting Information.