Difference between revisions of "Annealing primers"

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=Annealing complementary primers=
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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).
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#[[Annealing complementary primers]]<--For pieces of DNA shorter than the limit on primer length.
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#[[Annealing and primer extension]]<--For pieces of DNA longer than the limit on primer length.
  
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]
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[[Category:Protocol]]
 
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[[Category:DNA]]
*When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/&mu;l.
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[[Category:In vitro]]
 
 
*For the annealing mix one recipe that works is as follows -
 
**4 &mu;L of each of the concentrated primers.
 
**4 &mu;L of salt solution (10 mM NaCl)
 
**28 &mu;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&deg;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]]
 
 
 
=Annealing and primer extension=
 
 
 
This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp).  The DNA fragment is prepared for cloning by restriction digest.
 
 
 
==Materials==
 
 
 
*Two oligos which overlap by ~20 bp and have restriction enzyme sites at the 5' ends as in the diagram below.  See [[Restriction Digest]] for notes on cutting near the ends of linear DNA fragments.
 
 
 
Oligo 1: &nbsp;&nbsp;&nbsp;5' ---RE site-------------------------------- 3'<br>
 
Oligo 2:    &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3' --------------------------------RE site--- 5'
 
 
 
*[http://www.neb.com/nebecomm/products/productM0212.asp Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase]
 
*dNTPs (25 mM each dNTP in stock)
 
*Restriction enzyme(s)
 
*Restriction enzyme buffer
 
*BSA
 
 
 
==Calculating amount of oligo for reaction==
 
 
 
''This should be checked for errors'' -[[User:Rshetty|Reshma]] 19:03, 12 May 2005 (EDT)
 
 
 
<math> \rm{X\ L\ oligo} = \frac{\frac{Y\ g\ oligo}{(330\ g/mol\ of\ nt)(W\ nt/oligo)}\ mol\ of\ oligo}{Z\ mol/L\ oligo\ stock}</math>
 
 
 
==Procedure==
 
 
 
#Dilute the two oligos to a concentration of 10 or 25 &mu;M using H<sub>2</sub>O
 
#Mix the following in a 0.6 mL sterile tube
 
#*10 &mu;L 10X restriction enzyme buffer
 
#*1 &mu;L 100X BSA
 
#*X &mu;L oligo 1 (typically 1 &mu;g or more)
 
#*Y &mu;L oligo 1 (typically 1 &mu;g or more)
 
#*(87 - 2X) &mu;L deionized sterile H<sub>2</sub>O
 
#Anneal the two oligos together by either placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) at 94&deg;C for 5 mins followed by a cool down for 0.1&deg;C/sec to 37&deg;C or by placing the tube in a beaker of boiling water and letting it cool to room temperature.
 
#Add 1 &mu;L Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase to mixture. <br> Vortex polymerase before pipetting to ensure it is well-mixed.
 
#Add 1 &mu;L dNTPS (equal to 0.25 mM final concentration of each dNTP) <br> ''Recommend using a thermal cycler for the following incubation steps.''
 
#Incubate 1 hr at 37&deg;C.
 
#Heat inactivate polymerase by incubating at 75&deg;C for 20 minutes. <br> ''See [[Restriction Digest]] for more information on the following steps.''
 
#Add 1 &mu;L restriction enzyme(s) to mixture.
 
#Incubate for a minimum of 2 hrs.
 
#Heat inactivate restriction enzyme by incubating at 80&deg;C for 20 mins.
 
#[[Purification of DNA | Purify DNA]] as necessary
 

Latest revision as of 13:30, 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.