[Skip to Content](https://www.sigmaaldrich.com#main-content) [![MilliporeSigma](https://www.sigmaaldrich.com/static/logos/purple/millipore_sigma.svg)](https://www.sigmaaldrich.com/US/en) Products Cart0 USEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/US/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/US/en)[Polymerase Chain Reaction Applications](https://www.sigmaaldrich.com/US/en/applications/genomics/pcr)Standard PCR Protocol # Standard PCR Protocol ## How to do PCR A standard polymerase chain reaction (PCR) setup consists of four steps: 1. Add required reagents or mastermix and template to PCR tubes. 2. Mix and centrifuge. \*Add mineral oil to prevent evaporation in a thermal cycler without a heated lid. 3. Amplify per thermo cycler and primer parameters. 4. Evaluate amplified DNA by agarose gel electrophoresis followed by ethidium bromide staining. ![PCR tubes are loaded into a thermal cycler for the amplification step of PCR.](https://www.sigmaaldrich.com/content/dam/cms-commons/sigmaaldrich/marketing/global/images/technical-documents/protocols/genomics/pcr/loading_pcr_tubes_into_thermocycler.jpg "Loading PCR tubes into thermocycler") These steps are presented below in greater detail along with materials and reagent selection tips. This is a basic PCR protocol using Taq DNA polymerase. ## PCR Steps and Essential PCR Components Troubleshooting a PCR reaction involves many factors. Watch this animation to learn how selecting the right components and cycling conditions for your needs can help achieve optimum results. * * * Find additional protocols for other polymerases or advanced PCR techniques in the [Protocols](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/pcr-technologies-protocols-introduction) section of our PCR Technologies Guide. Learn more about standard PCR, including what it is, on our [PCR Basics page](https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/routine-amplification). ## [](https://www.sigmaaldrich.com)Reagents: What Is Needed for PCR? Reagent Used in PCRRecommended Product *Taq* DNA polymeraseSelect *Taq* DNA polymerase based upon user preference. (See separate [*Taq* polymerase table](https://www.sigmaaldrich.com#taqdnapolymerase) below.) PCR grade waterPCR Reagent Water Primers diluted to working concentration 10 µM working stocks are sufficient for most assays. Oligos[Custom oligos](https://www.sigmaaldrich.com/US/en/products/molecular-biology-and-functional-genomics/oligos-and-qpcr-probes/custom-predesigned-dna-oligos-and-qpcr-probes) DNA to be amplifiedProvided by researcher Dedicated pipettes Thermal cyclerWith various well block sizes or in multiformat Sterile filter pipette tips Sterile 1.5 mL screw-top microcentrifuge tubesCorning® microcentrifuge tubes with screw cap PCR tubes or platesChoose to fit cycler: Individual thin-walled 200 µL PCR tubes 200 µL strip tubes Multiwell plates and plate seal dNTP mixDeoxynucleotide mix containing 10 mM each of dATP, dCTP, dGTP, and dTTP \*readymixes already include dNTPs ## What is *Taq* Polymerase? *Taq* DNA polymerase is a thermostable enzyme derived from the thermophilic bacterium *Thermus aquaticus.* It is commonly used to amplify DNA fragments in PCR. The enzyme is in a recombinant form, expressed in *E. coli*. It is able to withstand repeated heating to 95 °C (as is demanded by the PCR technique) without significant loss of activity. The enzyme has a molecular weight of approximately 94 kDa by SDS-PAGE with no detectable endonuclease or exonuclease activity. It has 5'→3' DNA polymerase activity and 5'→3' exonuclease activity. Each lot of *Taq* DNA Polymerase is tested for PCR amplification and double-stranded sequencing. The enzyme is supplied at 5 units/µL and comes with an optimized 10x reaction buffer. [](https://www.sigmaaldrich.com) ### Standard *Taq* DNA Polymerase Use the table below to select an appropriate mix of *Taq* DNA polymerase for your reaction conditions. Choose from clear or red dyed formulations with and without magnesium chloride (MgCl2) or a pre-prepared readymix or master mix with buffer and dNTPs. Containing MgCl2 Separate MgCl2Readymix Clear formulation without dyeWith red dye for direct load on gelsClear formulation without dyeWith red dye for direct load on gelsClear formulation without dye *Taq* DNA Polymerase from *Thermus aquaticus* (D1806)RED*Taq*® DNA Polymerase (D4309)Taq DNA Polymerase from *Thermus aquaticus*, without MgCl2 (D4545)RED*Taq*® ReadyMix™ PCR Reaction Mix (R2523)ReadyMix™ *Taq* PCR Reaction Mix (P4600) __Unit Definition:__ One unit incorporates 10 nmol of total deoxyribonucleoside triphosphates into acid precipitable DNA in 30 minutes at 74 °C. ## [](https://www.sigmaaldrich.com)Procedure: Steps of PCR The optimal conditions for the concentration of *Taq* DNA polymerase, template DNA, primers, and MgCl2 will depend on the system being utilized. It may be necessary to determine the optimal conditions for each individual component. This is especially true for the *Taq* DNA polymerase, cycling parameters, and the MgCl2 concentration. It is recommended the enzyme and the MgCl2 be titrated to determine the optimal efficiency. 1. __Add the reagents to an appropriately sized tube__ in the order provided in the table. (Select appropriate table for reaction setup: standard or readymix reagent.) For a large number of reactions, a mastermix without the template should be set up and aliquoted into reaction tubes. At the end, template should be added to appropriate tubes. ### Standard PCR Reaction AmountComponentFinal Concentration w µLWater 5 µL10x PCR Buffer ([P2192](https://www.sigmaaldrich.com/US/en/product/sigma/P2192) or [P2317](https://www.sigmaaldrich.com/US/en/product/sigma/P2317))\*1x 1 µLDeoxynucleotide Mix200 µM w µLForward primer (typically 15-30 bases in length)0.1-0.5 µM x µL Reverse primer (typically 15-30 bases in length) 0.1-0.5 µM 0.5 µL *Taq* DNA Polymerase* 0.05 units/µL y µL Template DNA (typically 10 ng) 200 pg/µL z µL 25 mM MgCl2 (use only with buffer [P2317](https://www.sigmaaldrich.com/US/en/product/sigma/P2317)) 0.1-0.5 mM __50 µL__ __Total reaction volume__ \*Buy buffer and *Taq* polymerase together: D1806, D4309 or D4545 ### Readymix PCR Reaction AmountComponentFinal Concentration 25 µL Readymix ([R2523](https://www.sigmaaldrich.com/US/en/product/sigma/R2523) or [P4600](https://www.sigmaaldrich.com/US/en/product/sigma/P4600)) w µL Forward primer (typically 15-30 bases in length) 0.1-0.5 µM x µL Reverse primer (typically 15-30 bases in length) 0.1-0.5 µM y µL Template DNA (typically 10 ng) 200 pg/µL z µL Water __50 µL__ __Total reaction volume__ 2\. __Mix gently by vortex and briefly centrifuge__ to collect all components to the bottom of the tube. Note: Add 50 µL of mineral oil to the top of each tube to prevent evaporation if using a thermal cycler without a heated lid. 3\. __Amplify.__ The amplification parameters will vary depending on the primers and the thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler. ### Typical Cycling Parameters 25-30 cycles of amplification are recommended. | | | | |-------------------|----------------|----------| | PCR Step | Temperature °C | Duration | | Denature template | 94 °C | 1 min | | Anneal primers | 55 °C | 2 min | | Extension | 72 °C | 3 min | 4\. __The amplified DNA can be evaluated by [agarose gel electrophoresis](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/nucleic-acid-gel-electrophoresis/nucleic-acid-electrophoresis) and subsequent ethidium bromide staining.__ Note: Mineral oil overlay may be removed by a single chloroform extraction (1:1), recovering the aqueous phase. ### [](https://www.sigmaaldrich.com)Reagents for Nucleic Acid Electrophoresis - Agarose (precast gels, powder, etc.) - Buffer such as MOPS-EDTA-sodium acetate, tris-acetate-EDTA (TAE) or tris-borate-EDTA (TBE) - Gel loading solution and sample loading buffer for RNA - Electrophoresis stain or dye such as ethidium bromide 1\. Cheng S, Fockler C, Barnes WM, Higuchi R. 1994. Effective amplification of long targets from cloned inserts and human genomic DNA.. Proceedings of the National Academy of Sciences. 91(12):5695-5699. [https://doi.org/10.1073/pnas.91.12.5695](https://doi.org/10.1073/pnas.91.12.5695) 2\. Chou Q. 1992. Minimizing deletion mutagenesis artifact duringTaqDNA polymerase PCR byE.coliSSB. Nucl Acids Res. 20(16):4371-4371. [https://doi.org/10.1093/nar/20.16.4371](https://doi.org/10.1093/nar/20.16.4371) 3\. Innis MA. 1995. PCR Strategies. New York: Academic Press. 4\. Innis MA. 1990. PCR Protocols: A Guide to Methods and Applications. New York: Academic Press. 5\. Innis MA, Myambo KB, Gelfand DH, Brow MA. 1988. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA.. Proceedings of the National Academy of Sciences. 85(24):9436-9440. [https://doi.org/10.1073/pnas.85.24.9436](https://doi.org/10.1073/pnas.85.24.9436) 6\. Newton CR. 1995. PCR: Essential Data. 1. Wiley-Blackwell. 7\. Olive DM, Simsek M, Al-Mufti S. 1989. Polymerase chain reaction assay for detection of human cytomegalovirus.. 27(6):1238-1242. [https://doi.org/10.1128/jcm.27.6.1238-1242.1989](https://doi.org/10.1128/jcm.27.6.1238-1242.1989) 8\. Pääbo S, Gifford JA, Wilson AC. 1988. Mitochondrial DNA sequences from a 7000-year old brain. Nucl Acids Res. 16(20):9775-9787. [https://doi.org/10.1093/nar/16.20.9775](https://doi.org/10.1093/nar/16.20.9775) 9\. Erlich HA. 1989. PCR technology : principles and applications for DNA amplification. New York: Stockton Press. 10\. Sambrook J, Fritsch E, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 1. New York: Cold Spring Harbor Laboratory Press. 11\. Sarkar G, Kapelner S, Sommer SS. 1990. Formamide can dramatically improve the specificity of PCR. Nucl Acids Res. 18(24):7465-7465. [https://doi.org/10.1093/nar/18.24.7465](https://doi.org/10.1093/nar/18.24.7465) 12\. Winship PR. 1989. An lmproved method for directly sequencing PCR-amplified material using dimethyl sulphoxide. Nucl Acids Res. 17(3):1266-1266. [https://doi.org/10.1093/nar/17.3.1266](https://doi.org/10.1093/nar/17.3.1266) ## Label License Statement ### NOTICE TO PURCHASER: DISCLAIMER OF LICENSE No license is conveyed with the purchase of this product under any of US Patents Nos. 5,804,375, 5,994,056, 6,171,785, 6,214,979, 5,538,848, 5,723,591, 5,876,930, 6,030,787, and 6,258,569, and corresponding patents outside the United States, or any other patents or patent applications, relating to the 5’ Nuclease and dsDNA-Binding Dye Processes. For further information contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA ## Materials Sorry, an unexpected error has occurred Response not successful: Received status code 500 __Related Articles__ - [Installation & Use of Milli-Q® SQ *2Series* Water Purification Systems](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/wet-chemical-analysis/installation-use-of-milli-q-2series-water-purification-system) - [Optimized PCR-based Detection of Mycoplasma](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-counting-and-health-analysis/optimized-pcr-based-mycoplasma-detection) - [Recombinant Selection](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/cloning-and-expression/restriction-enzyme-cloning-manual-recombinants) - [Whole Genome Amplification: Blood Card Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/dna-and-rna-purification/blood-card) - [Deoxyribonuclease I](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/dna-and-rna-purification/deoxyribonuclease-i) - [Extract-N-Amp™ Tissue PCR Kit Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/dna-and-rna-purification/extract-n-amp-tissue-pcr-kit) - [Saliva DNA Extraction & WGA Amplification](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/dna-and-rna-purification/extraction-of-dna-from-saliva) - [DNA Extraction & WGA Amplification from Soil Samples](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/genomics/dna-and-rna-purification/extraction-of-dna-from-soil) - [View More](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) __Related Product Categories__ - [Sealing Films, Foils & Tapes](https://www.sigmaaldrich.com/US/en/products/labware/sealing-films-foils-and-tapes) - [Allergenic Fragrance Testing – Certified Materials](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/allergenic-fragrance-testing) - [Analysis of Alkylphenols Using New Internal Standards](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/analysis-of-alkylphenols) - [FAME Standard for Optimizing GC System Performance](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/fame-standard-for) - [Fast and Reliable Environmental Analysis of Aldehyde and Ketone Air Pollutants](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/fast-and-reliable) - [Hydrocarbon Oil Index in Samples](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/hydrocarbon-oil-index) - [Neonicotinoids](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/neonicotinoids-bee-pesticides) - [New GC-MS Method for Mycotoxin Analysis](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/new-gc-ms-method-for-mycotoxin-analysis) - [View More](https://www.sigmaaldrich.com/US/en/search/facet-search?focus=sitecontent&term=facet-search) Top __Sign In To Continue__ To continue reading please sign in or create an account. 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