Although it may be possible to anneal oligos at room temperature, heating to denature the oligos and then cooling slowly to anneal the two oligos will ensure more efficient annealing and favor the most stable duplex formation. If you choose not to heat the oligos, it would be prudent to carefully screen the oligos for secondary structures which could interfere with the annealing reaction. ANNEALING PROTOCOL
: Dissolve the oligos at high concentration—as high as 500 µM if possible, although as low as 100 µM will work; i.e., 1-10 OD260 units / 100 µL—in STE Buffer (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA) or Nuclease-free Duplex Buffer (30 mM Hepes pH 7.5, 100 mM KAc) (available from IDT). The presence of some salt is necessary for the oligos to hybridize. Then mix the two oligos in equimolar concentrations, heat to 94° C, and allow the solution to cool slowly to room temperature. For sequences with significant hairpin potential, a more gradual cooling step is beneficial; this is easily done by placing the oligos in a water bath or heating block and unplugging the machine. The resulting product will be a stable, double-stranded construct.