Annealing primers

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Procedure)
Current revision (17:30, 9 October 2007) (view source)
m (Annealing Primers moved to Annealing primers: case convention)
 
(7 intermediate revisions not shown.)
Line 1: Line 1:
-
==Annealing complementary primers==
+
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.
-
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]
+
[[Category:Protocol]]
-
 
+
[[Category:DNA]]
-
*When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/&mu;l.
+
[[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 2 (typically 1 &mu;g or more)
+
-
#*(87 - X - Y) &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, a cool down for 0.1&deg;C/sec to 65&deg;C, 65&deg;C for 5 mins, then a cool down for 0.1&deg;C/sec to 37&deg;C.  Alternatively, the tube can be placed in a beaker of boiling water and let 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
+
-
 
+
-
==References==
+
-
 
+
-
W. P. Stemmer, A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene, 164(1):49–53, 1995. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=7590320&dopt=Abstract PubMed]
+

Current revision

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.
Personal tools