What is your protocol for annealing oligos?

Mar 21, 2014, 14:29 PM

It is sometimes necessary to make double-stranded DNA from single-stranded oligos. While the annealing procedure is simple, attention to a few details can greatly reduce the presence of undesired single-stranded material.


Dissolve each oligo in “Duplex Buffer” (100 mM Potassium Acetate, 30 mM HEPES, pH 7.5) at high concentration (1−10 OD260 units/100 μL). The presence of some salt is necessary for the oligos to hybridize.

  1. Mix the two sequences together in equal molar amounts. If different amounts are used, there will always be single-stranded sequence left over.
  2. Heat to 94°C and cool gradually. For many oligos this can be as simple as transferring from 94°C to the bench-top (room temperature). For sequences with significant hairpin potential, a more gradual cooling/annealing step is beneficial; this is easily done by placing the oligos in a water bath or heat block and unplugging the machine.
  3. The resulting product will be in stable, double-stranded form and can be stored at 4°C or frozen.

Additional tips:

If the product will be used in a ligation reaction, the addition of 5’-phosphate may be needed. This can be done at the time of oligo synthesis (chemical phosphorylation) or at any time thereafter using PNK (enzymatic phosphorylation). If the oligos are relatively long or will be used in cloning, starting with PAGE-purified oligos is recommended.

  • Annealing
  • protocol
  • annealing
  • Duplex