Endy:Annealing complementary primers

See also Annealing complementary primers

Primer reconstitution

When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/μl. See this page on reconstituting primers for more information.

Annealing Mix

• For the annealing mix one recipe that works is as follows -
  • 8 μL of each of the concentrated primers.
  • 4 μL of salt solution (10 mM NaCl)
  • 20 μL of water

Option 1

Anneal the primers by heating them at least 5°C above their melting point and cooling them down slowly in stages using a Thermocycler. Melting temperature calculations can best be done using software such as VectorNTI or data may come with the primers themselves.

Option 2

A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally. Most primers pairs with length less than 100bp should be fully melted at 100^oC and certainly any non-specific binding should be melted at that temperature.