Standard PCR Protocol

researcher loading PCR tubes into a thermocycler description=

Overview: 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.

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

 

Our interactive PCR Selection Guide allows you to sort master mixes and other PCR-specific reagents based on your needs or reaction type.

 

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
  1. 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.
  2. 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
  1. 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

References

  1. Cheng, S., et al.,  Proc. Natl. Acad. Sci. USA, 91, 5695-5699 (1994).
  2. Chou, Q., Nucleic Acids Res. 20, 4371 (1992).
  3. Innis, M.A., et al. (Eds.)  PCR Strategies, Academic Press, New York (1995).
  4. Innis, M., et al. (Eds.)  PCR Protocols:  A Guide to Methods and Applications, Academic Press, San Diego, California (1990).
  5. Innis, M., et al., Proc. Natl. Acad. Sci. USA 85, 9436-9440 (1988).
  6. Newton, C.R.  (Ed.)  PCR: Essential Data, John Wiley & Sons, New York (1995).
  7. Olive, D., et al., J. Clin. Microbiol. USA 27, 1238 (1989).
  8. Paabo, S., et al., Nucleic Acids Res. 16, 9775-9787 (1988).
  9. Saiki, R., PCR Technology: Principles and Applications for DNA Amplification, Stockton, New York (1989).
  10. Sambrook, J, et al.  Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, New York (2000) . Catalog No. M8265.
  11. Sarkar, G., et al., Nucleic Acids Res. 18, 7465 (1990).
  12. Winship, P.R., et al., Nucleic Acids Res. 17, 1266 (1989).

 

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

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