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| - | ==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. |
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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].
| + | [[Category:Protocol]] |
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| - | *When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/μl.
| + | [[Category:In vitro]] |
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| - | *For the annealing mix one recipe that works is as follows -
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| - | **4 μL of each of the concentrated primers.
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| - | **4 μL of salt solution (10 mM NaCl)
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| - | **28 μL of water
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| - | *The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind.
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| - | *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.
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| - | *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.
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| - | *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]]
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| - | ==Annealing and primer extension==
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| - | 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.
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| - | ===Materials===
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| - | *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.
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| - | Oligo 1: 5' ---RE site-------------------------------- 3'<br>
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| - | Oligo 2: 3' --------------------------------RE site--- 5'
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| - | *[http://www.neb.com/nebecomm/products/productM0212.asp Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase]
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| - | *dNTPs (25 mM each dNTP in stock)
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| - | *Restriction enzyme(s)
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| - | *Restriction enzyme buffer
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| - | *BSA
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| - | ===Calculating amount of oligo for reaction===
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| - | ''This should be checked for errors'' -[[User:Rshetty|Reshma]] 19:03, 12 May 2005 (EDT)
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| - | <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>
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| - | ===Procedure===
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| - | #Dilute the two oligos to a concentration of 10 or 25 μM using H<sub>2</sub>O
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| - | #Mix the following in a 0.6 mL sterile tube
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| - | #*10 μL 10X restriction enzyme buffer
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| - | #*1 μL 100X BSA
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| - | #*X μL oligo 1 (typically 1 μg or more)
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| - | #*Y μL oligo 2 (typically 1 μg or more)
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| - | #*(87 - X - Y) μL deionized sterile H<sub>2</sub>O
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| - | #Anneal the two oligos together by either placing the mixture in a thermal cycler ([http://www.mjr.com MJ Research], PTC-200) at 94°C for 5 mins, a cool down for 0.1°C/sec to 65°C, 65°C for 5 mins, then a cool down for 0.1°C/sec to 37°C. Alternatively, the tube can be placed in a beaker of boiling water and let cool to room temperature.
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| - | #Add 1 μL Klenow 3'<math>\rightarrow</math>5' exo<sup>-</sup> polymerase to mixture. <br> Vortex polymerase before pipetting to ensure it is well-mixed.
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| - | #Add 1 μL dNTPS (equal to 0.25 mM final concentration of each dNTP). <br> ''Recommend using a thermal cycler for the following incubation steps.''
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| - | #Incubate 1 hr at 37°C.
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| - | #Heat inactivate polymerase by incubating at 75°C for 20 minutes. <br> ''See [[Restriction Digest]] for more information on the following steps.''
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| - | #Add 1 μL restriction enzyme(s) to mixture.
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| - | #Incubate for a minimum of 2 hrs.
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| - | #Heat inactivate restriction enzyme by incubating at 80°C for 20 mins.
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| - | #[[Purification of DNA | Purify DNA]] as necessary
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| - | ==References==
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| - | 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]
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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 part fabrication for other ways to make a part (contains BioBrick specific details).
