Annealing primers

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Current revision (17:30, 9 October 2007) (view source)
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A simple and cheap way to make a short (<100bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen].
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Annealing primers can be used as a fast and cheap way to synthesize a short piece of DNA for which you do not have template DNA to PCR from.  See [[Synthetic Biology:BioBricks/Part fabrication|part fabrication]] for other ways to make a part (contains [[Synthetic Biology:BioBricks|BioBrick]] specific details).
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#[[Annealing complementary primers]]<--For pieces of DNA shorter than the limit on primer length.
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#[[Annealing and primer extension]]<--For pieces of DNA longer than the limit on primer length.
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*When the primers arrive, redissolve them in 50mM Tris buffer to yield a concentration of ~800ng/<math>\mu</math>l.
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[[Category:Protocol]]
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[[Category:DNA]]
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*For the annealing mix one recipe that works is as follows -
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[[Category:In vitro]]
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**4<math>\mu</math>l of each of the concentrated primers.
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**4<math>\mu</math>l of salt solution (10mM NaCl)
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**28<math>\mu</math>l of water
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*The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.
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*Anneal the primers by heating them at least 5<sup>o</sup>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.
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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.
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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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Current revision

Annealing primers can be used as a fast and cheap way to synthesize a short piece of DNA for which you do not have template DNA to PCR from. See part fabrication for other ways to make a part (contains BioBrick specific details).

  1. Annealing complementary primers<--For pieces of DNA shorter than the limit on primer length.
  2. Annealing and primer extension<--For pieces of DNA longer than the limit on primer length.
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