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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]] |
| | | [[Category:DNA]] |
| *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 1 (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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