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The page below is a sample from the LabCE course PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Optimization of dNTPs, MgCl2, Polymerase, and Salts

Optimize amounts of dNTPs, MgCl2, polymerase, and salts. When running so many reactions at once, it is important to have extra dNTPs and magnesium so the reaction does not run out of components.

As mentioned earlier, dNTPs are the nucleotide bases added to the growing DNA strand by the DNA polymerase. The concentration of each dNTP in the reaction mixture is usually 200 μM. It is very important to have equal concentrations of each dNTP (dATP, dCTP, dGTP, dTTP), as inaccuracy in the concentration of a single dNTP
dramatically increases the opportunity for misincorporation.

Taq DNA polymerase - This DNA polymerase is isolated from the bacterium Thermus aquaticus, which lives in hot environments and maintains biomolecules that are heat stable. Therefore, Taq DNA polymerase can efficiently synthesize DNA under the heat intensive conditions of the PCR reaction. Note that there are other polymerases, but Taq is the most commonly used. The ideal temperature for Taq DNA polymerase activity is 72°C. Lower temperatures may predispose to non-specific binding, while higher temperatures may be too stringent.

Usually, 0.02 units of Taq DNA polymerase are used per μl of reaction mix. Higher Taq DNA polymerase concentrations may cause synthesis of nonspecific products. However, if inhibitors are present in the reaction mix (eg, if the template DNA used is not highly purified), higher amounts of Taq DNA polymerase (0.04-0.06 units) may be necessary to obtain a better yield of amplification products.

MgCl2 - The concentration of MgCl2 influences the stringency of the interaction between the primers and the template DNA. The range of MgCl2 usually is from 0.5 - 4 mM
  • Low MgCl2 concentrations can help to eliminate non-specific priming and background PCR products and are desirable when fidelity of DNA synthesis is critical. At the same time however, too few Mg2+ ions can result in a low yield of PCR product.
  • High MgCl2 concentrations can help to stabilize interaction of the primers with their intended template if it is not being amplified, but can also result in nonspecific binding and erroneous PCR product formation. The amount of additional MgCl2 must be carefully monitored.
Salt and buffers - Stabilize enzyme function and formation of primer-template hybrids. Buffers often contain Tris-HCl, KCl, and sometimes MgCl2. Occasionally bovine serum albumin is added to enhance yield.