Endy:Annealing complementary primers

From OpenWetWare
Revision as of 11:10, 10 May 2007 by Barry Canton (talk | contribs) (Annealing Mix)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to: navigation, search

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. Consider doing a PNK step on the primers if they do not have 5' phosphates and you intend to use them for ligation.

Annealing Mix

This reaction mix and multiples thereof are known to work but they have not been extensively optimized so improvements likely exist.

  • 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 primer pairs with length less than 100bp should be fully melted at 100oC and certainly any non-specific binding should be melted at that temperature.