Annealing of siRNAs
Biosynthesis can deliver siRNA as annealed duplexes. However, To ensure maximum stability, we usually ship modified RNA lyophilized in individual tubes. If you require annealing, use the following protocol.
RNA oligonucleotides are highly susceptible to degradation by exogenous RNases introduced during handling. Therefore, perform all handling steps under sterile, RNase-free conditions.
Upon receipt, dried RNA oligonucleotides may be safely stored in a freezer for up to 6 months at -20°C.
Separately aliquot and dilute each RNA oligo to a concentration of 50 µM. Combine 30 µl of each RNA oligo solution and 15 µl of 5x annealing buffer (see below). The final volume is 75 µl. The final concentration of the duplex is 20 µM.
Incubate the solution for 1-2 minutes in a water bath at 90-95°C, and allow it to cool to room temperature on your workbench. Slow cooling should take about 45-60 minutes. Centrifuge the tube briefly to collect all liquid at the bottom of the reaction tube. Store on ice until ready to use.
Once annealed, duplex siRNA is much more resistant to nuclease than single-stranded RNA and can be safely stored frozen at -20°C. The 5x annealing buffer can be freeze-thawed up to 5 times.
5x annealing buffer
50 mM Tris, pH 7.5
100 mM NaCl