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.

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 section of our PCR Technologies Guide.

Learn more about standard PCR, including what it is, on our PCR Basics page.

Reagents: What Is Needed for PCR?

Reagent Used in PCR	Recommended Product
Taq DNA polymerase	Select Taq DNA polymerase based upon user preference. (See separate Taq polymerase table below.)
PCR grade water	PCR Reagent Water
Primers diluted to working concentration	10 µM working stocks are sufficient for most assays.
Oligos	Custom oligos
DNA to be amplified	Provided by researcher
Dedicated pipettes
Thermal cycler	With various well block sizes or in multiformat
Sterile filter pipette tips
Sterile 1.5 mL screw-top microcentrifuge tubes	Corning® microcentrifuge tubes with screw cap
PCR tubes or plates	Choose to fit cycler:
	Individual thin-walled 200 µL PCR tubes
	200 µL strip tubes
	Multiwell plates and plate seal
dNTP mix	Deoxynucleotide 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.

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 MgCl2	Readymix
Clear formulation without dye	With red dye for direct load on gels	Clear formulation without dye	With red dye for direct load on gels	Clear formulation without dye
Taq DNA Polymerase from Thermus aquaticus (D1806)	REDTaq® DNA Polymerase (D4309)	Taq DNA Polymerase from Thermus aquaticus, without MgCl2 (D4545)	REDTaq® 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.

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

Amount	Component	Final Concentration
w µL	Water
5 µL	10x PCR Buffer (P2192 or P2317)*	1x
1 µL	Deoxynucleotide Mix	200 µM
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
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)	0.1-0.5 mM
50 µL	Total reaction volume

*Buy buffer and Taq polymerase together: D1806, D4309 or D4545

Readymix PCR Reaction

Amount	Component	Final Concentration
25 µL	Readymix (R2523 or 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 and subsequent ethidium bromide staining.

Note: Mineral oil overlay may be removed by a single chloroform extraction (1:1), recovering the aqueous phase.

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

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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

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

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

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

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

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

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