Annealing primers

A simple and cheap way to make a short (<100bp) piece of DNA is to order two complementary primers from a company such as Invitrogen.

• When the primers arrive, redissolve them in 50mM Tris buffer to yield a concentration of ~800ng/[math]\displaystyle{ \mu }[/math]l.

• For the annealing mix one recipe that works is as follows -
  • 4[math]\displaystyle{ \mu }[/math]l of each of the concentrated primers.
  • 4[math]\displaystyle{ \mu }[/math]l of salt solution (10mM NaCl)
  • 28[math]\displaystyle{ \mu }[/math]l of water

• The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.

• Anneal the primers by heating them at least 5^oC 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.

• 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.

• Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by using adding the 5' phosphate using a protocol such as PNK Treatment of DNA Ends