Koch Lab:Protocols/Oligonucleotide Annealing/Duplexes

From OpenWetWare
Jump to navigationJump to search

Often, two complementary oligos are synthesized separately, and one wants to create duplex dsDNA by hyrbidizing the oligos. If you simply mix them together, it's possible that there will be stable hairpin structures that take a very long time (at room temp) to unpair. Therefore, an annealing process is used: heat the oligo mixture and gradually cool. The gradual cooling is likely to allow the oligos to find the "correct" lowest energy configuration at room temperature. This process could also be used for a fairly long hairpin, or three or more oligos.

Related OWW content

  • Annealing_complementary_primers The OWW protocols are for annealing and primer extension, whereas our Koch Lab protocols tend to be for annealing and ligation (for single-molecule tethering).

General protocol

  1. Mix oligos together at high concentration in a buffer with reasonable ionic strength (common is TE + 50 mM NaCl).
  2. Heat oligo mixture to 95C (close to boiling).
  3. Cool down gradually to room temperature (the level of "gradualness" required depends on how worried you are about other stable structures). One way to make it gradual is to program a thermal cycler (e.g. 0.5 degree / minute). Another way is to use a large thermal mass, such as a beaker of boiling water.
  4. Freeze for storage (no need to gradually freeze).

Assessing products

There is no easy method to assess whether the "correct" duplex has been formed. There are probably many difficult methods (for example, native PAGE with SYBR stain). So, usually you just hope (e.g., if the next step works reasonably well, you assume it's OK).


  • Salt is necessary or at least helps, because oligos are highly charged.