Annealing primers: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
 
m (Annealing Primers moved to Annealing primers: case convention)
 
(15 intermediate revisions by 3 users not shown)
Line 1: Line 1:
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].
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).
#[[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.


*When the primers arrive, redissolve them in 50mM Tris buffer to yield a concentration of ~800ng/<math>\mu</math>l.
[[Category:Protocol]]
 
[[Category:DNA]]
*For the annealing mix one recipe that works is as follows -
[[Category:In vitro]]
**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 Primers]]

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).

  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.
Retrieved from "https://openwetware.org/mediawiki/index.php?title=Annealing_primers&oldid=157216"