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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]. |+|
and cheap way to a short piece of DNA to froma [:.
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|−|*When the primers arrive, redissolve them in 50mM Tris buffer to yield a concentration of ~800ng/<math>\mu</math>l. |+|
|−|*For the annealing mix one recipe that works is as follows - |+|
|−|**4<math>\mu</math>l of each of the concentrated primers. | |
|−|**4<math>\mu</math>l of salt solution (10mM NaCl) | |
|−|**28<math>\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<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. | |
|−|*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. | |
|−|*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]] | |
Latest revision as of 14:30, 9 October 2007
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).
- Annealing complementary primers<--For pieces of DNA shorter than the limit on primer length.
- Annealing and primer extension<--For pieces of DNA longer than the limit on primer length.