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| [[Endy:Annealing complementary primers]]<br> | | [[Endy:Annealing complementary primers]]<br> |
| [[Silver: Oligonucleotide Inserts|Silver:Annealing complementary primers]]<br> | | [[Silver: Oligonucleotide Inserts|Silver:Annealing complementary primers]]<br> |
| ==Method==
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| ===Primer reconstitution===
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| 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 | reconstituting primers]] for more information.
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| ===Annealing Mix===
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| *For the annealing mix one recipe that works is as follows -
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| **8 μL of each of the concentrated primers.
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| **4 μL of salt solution (10 mM NaCl)
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| **20 μL of water
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| ===Option 1===
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| Anneal the primers by heating them at least 5°C above their melting point and cooling them down slowly in stages using a [[Endy:Thermocyclers|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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| ===Option 2===
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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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| ==Notes== | | ==Notes== |
