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General Handling Instructions
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.
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