Annealing primers

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*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<sup>o</sup>C and certainly any non-specific binding should be melted at that temperature.
*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<sup>o</sup>C and certainly any non-specific binding should be melted at that temperature.
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*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 Primers]]
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*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]]

Revision as of 19:32, 3 May 2005

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/μl.
  • For the annealing mix one recipe that works is as follows -
    • 4μl of each of the concentrated primers.
    • 4μl of salt solution (10mM NaCl)
    • 28μl 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 5oC 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 100oC 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
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