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Protocol
Basic PCR protocol
Keywords: PCR, polymerase chain reaction, amplification, Taq
Amplify a DNA template using oligonucleotide primers.
The polymerase chain reaction (PCR) allows exponential amplification of small amounts of DNA template in vitro. A typical reaction includes the target DNA, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium. Once assembled, the reaction is placed in a thermal cycler and subjected to multiple cycles of template denaturation, primer annealing and primer extension. Each PCR cycle theoretically doubles the amount of target sequence in the reaction so that, at the end of 20 cycles, the DNA template is amplified by factor of more than a million in a matter of hours.
Required Materials
- template DNA
- downstream PCR primer
- upstream PCR primer
- GoTaq® DNA Polymerase
- 5X Colorless GoTaq® Flexi Buffer , or Green GoTaq, supplied with GoTaq® DNA Polymerase
- MgCl2 , 25 mM, supplied with GoTaq® DNA Polymerase
- RNase free water
- nuclease-free light mineral oil , if using a thermal cycler without a heated lid
- dNTP kit (10 mM) , either home made or purchased dNTP mix
Main steps
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Combine the first five reaction components in the order listed below in…
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Overlay the reaction with 1–2 drops (20–40μl) of nuclease-free mineral…
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Place the tubes in a thermal cycler, and proceed with the thermal cycling…
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Analyze 5μl of the PCR products by agarose gel electrophoresis. The products…
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Store reaction products at –20°C until needed.
Instructions
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Combine the first five reaction components in the order listed below in a thin-walled 0.5ml reaction tube. Gently vortex the tube for 10 seconds, and briefly centrifuge in a microcentrifuge. Initiate the reaction by adding the template and primers.
Component Volume Final Concentration Nuclease-Free Water (to a final volume of 50μl) Xμl 5X Green or Colorless GoTaq® Flexi Buffer 10μl 1X dNTP mix, 10mM each dNTP 1μl 0.2mM each GoTaq® DNA Polymerase (5u/μl) 0.25μl 0.025unit/μl 25mM MgCl2 3μl 1.5mM Downstream primer 50pmol 1μM Upstream primer 50pmol 1μM Template DNA* Yμl *If possible, start with >104 copies of the target sequence to obtain a signal in 25–30 cycles, but keep the final DNA concentration of the reaction at ≤ 10ng/μl.
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Overlay the reaction with 1–2 drops (20–40μl) of nuclease-free mineral oil to prevent condensation and evaporation. Mineral oil addition is not necessary if you are using a thermal cycler with a heated lid.
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Place the tubes in a thermal cycler, and proceed with the thermal cycling profile determined to be optimal for your reactions.
Note: Information about PCR optimization can be found at: http://www.promega.com/paguide/chap1.htm
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Analyze 5μl of the PCR products by agarose gel electrophoresis. The products should be readily visible by UV transillumination of the ethidium bromide-stained gel.
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Store reaction products at –20°C until needed.
