Annealing complementary primers: Difference between revisions

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A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen]. 
''See also [[Annealing primers]]''


*When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/&mu;l.
==Introduction==
A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen].  Other options are described under [[Synthetic Biology:BioBricks/Part fabrication | part fabrication]] and [[Annealing and primer extension|annealing and primer extension]].  Having annealed the primers, you most likely want to use them as an insert into a [[Synthetic Biology:Vectors | vector]] (the linked page describes BioBrick vectors).  Here are some pages that describe methods to assemble two parts if they correspond to a BioBricks standard -
#[[BioBricks construction tutorial]]
#[[Synthetic Biology:BioBricks/3A assembly | 3A assembly]]
#[[Silver: BB Strategy | Silver lab strategy]].


*For the annealing mix one recipe that works is as follows -
==Lab Protocols==
**8 &mu;L of each of the concentrated primers.
[[Endy:Annealing complementary primers]]<br>
**4 &mu;L of salt solution (10 mM NaCl)
[[Silver: Oligonucleotide Inserts|Silver:Annealing complementary primers]]<br>
**20 &mu;L of water
[[Koch Lab:Protocols/Oligonucleotide Annealing/Duplexes|Koch Lab]] (For single-molecule tethering purposes.)<br>


*The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.
==Notes==
*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]


*Anneal the primers by heating them at least 5&deg;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.
*The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.  The salt concentration listed above is designed to give an excess of positive sodium ions over negatively charged DNA bases.


*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.
*As the paired oligos cool, they will also form multimers of your insert.  To release them, you should heat the mixture of your vector and insert DNA to about 65C and let it cool prior to adding ligase.


*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]]
*If you will subsequently be ligating the annealed primers into a vector, it is possible to add the vector into the annealing mix and then add ligase buffer and ligase once the mix gets close to room temperature.  this should reduce the likelihood of insert multimers forming.

Latest revision as of 16:36, 7 June 2008

See also Annealing primers

Introduction

A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as Invitrogen. Other options are described under part fabrication and annealing and primer extension. Having annealed the primers, you most likely want to use them as an insert into a vector (the linked page describes BioBrick vectors). Here are some pages that describe methods to assemble two parts if they correspond to a BioBricks standard -

  1. BioBricks construction tutorial
  2. 3A assembly
  3. Silver lab strategy.

Lab Protocols

Endy:Annealing complementary primers
Silver:Annealing complementary primers
Koch Lab (For single-molecule tethering purposes.)

Notes

  • Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by adding the 5' phosphate using a protocol such as PNK Treatment of DNA Ends
  • The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind. The salt concentration listed above is designed to give an excess of positive sodium ions over negatively charged DNA bases.
  • As the paired oligos cool, they will also form multimers of your insert. To release them, you should heat the mixture of your vector and insert DNA to about 65C and let it cool prior to adding ligase.
  • If you will subsequently be ligating the annealed primers into a vector, it is possible to add the vector into the annealing mix and then add ligase buffer and ligase once the mix gets close to room temperature. this should reduce the likelihood of insert multimers forming.
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