Endy:Annealing complementary primers: Difference between revisions
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''See also [[Annealing complementary primers]]'' | ''See also [[Annealing complementary primers]]'' | ||
==Primer reconstitution== | ==Primer reconstitution== | ||
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. | 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. Consider doing a [[PNK Treatment of DNA Ends|PNK step]] on the primers if they do not have 5' phosphates and you intend to use them for ligation. | ||
==Annealing Mix== | ==Annealing Mix== | ||
This reaction mix and multiples thereof are known to work but they have not been extensively optimized so improvements likely exist. | |||
*8 μL of each of the concentrated primers | |||
*4 μL of salt solution (10 mM NaCl) | |||
*20 μL of water | |||
==Option 1== | ==Option 1== | ||
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==Option 2== | ==Option 2== | ||
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 | 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 primer 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. | ||
Latest revision as of 12:10, 10 May 2007
See also Annealing complementary primers
Primer reconstitution
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 for more information. Consider doing a PNK step on the primers if they do not have 5' phosphates and you intend to use them for ligation.
Annealing Mix
This reaction mix and multiples thereof are known to work but they have not been extensively optimized so improvements likely exist.
- 8 μL of each of the concentrated primers
- 4 μL of salt solution (10 mM NaCl)
- 20 μL of water
Option 1
Anneal the primers by heating them at least 5°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.
Option 2
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 primer pairs with length less than 100bp should be fully melted at 100oC and certainly any non-specific binding should be melted at that temperature.
