MgCl2 (25mM)

Magnesium ion (Mg2+) is an essential cofactor for DNA polymerase activity, as it enables the incorporation of dNTPs during DNA polymerization. Moreover, Mg2+ facilitates the binding of primers to DNA templates by stabilizing negative charges, thereby influencing annealing and melting temperatures.

High Mg2+ concentrations increase polymerase activity, however, at the cost of fidelity. dsDNA is stabilized by elevated Mg2+ concentrations thereby preventing denaturation and increasing the melting temperature. For the same reason, primer-dimer formation is increased with higher Mg2+ concentrations as well as annealing temperatures.

Bioavailability of Mg2+ in a PCR reaction is dependent on many parameters such as template DNA concentration and purity, primer concentration and purity, dNTP concentration, presence of chelators (e.g. EDTA) or the presence, type and concentration of fluorescent dyes. Thus, it is recommended to optimize the Mg2+ concentration for each new PCR reaction or whenever changing these parameters.