All reagents used in the synthesis and purification of Ambion's siRNA are tested and certified nuclease-free. However, RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during handling. As a precaution, your RNA oligonucleotides should not be handled with ungloved hands. RNase-free reagents, pipette tips, and tubes should be used. Upon receipt, dried RNA oligonucleotides may be safely stored in a non-frost free freezer for 6 months at -20°C.
Resuspension of Single-Stranded siRNA Oligonucleotides
Briefly centrifuge tubes to ensure that the dried oligonucleotide is at the bottom of the tube. Resuspend oligonucleotides at a convenient concentration, e.g. 100 µM, in the RNase-free water provided. Store at -20°C.
Annealing of siRNA
Ambion provides 5X Annealing Buffer with each siRNA. In an RNase-free microfuge tube, combine the sense and antisense RNA oligonucleotides, water, and 5X siRNA Annealing Buffer. The final concentration should be 20 µM for each oligonucleotide and 1X Annealing Buffer. Heat for 1 min at 90¨C in a preheated thermal cycler or heat block, followed by incubation for 1 hour at 37¨C. Once annealed, double stranded siRNA is much more nuclease resistant than single-stranded RNA and can be safely stored frozen at -20¨C in a non-frost free freezer.