Magnesium is a required cofactor for thermostable DNA polymerases. Mg²⁺ in the PCR mixture stabilizes dsDNA and raises the Tm. Mg²⁺ concentration therefore is an important for controlling the specificity of the reaction. A low Mg²⁺ concentration requires more stringent base pairing in the annealing step. Too few Mg²⁺ ions result in a low yield of PCR product; too many Mg²⁺ ions increase the yield of non-specific products and promote misincorporation.

Insufficient Mg²⁺ concentration in a PCR mixture can causes failure of the reaction. Excess magnesium (or the presence of manganese) will cause the fidelity of DNA polymerases to be reduced and may cause the generation of unwanted products. On a gel this can appear as a ladder or smear. The MgCl₂ concentration should normally be between 1mM and 4mM. Since dNTPs sequester Mg²⁺ ions, a major change in the dNTP concentration in a rection would require a change in the concentration of MgCl₂. Similarly, changing the KCl-based buffer concentration or any other component of the PCR mix may require adjustment of the Mg²⁺ concentration in the reaction mixture.

Further reading:

1. PCR Books
2. Real-Time PCR: Current Technology and Applications
3. Real-Time PCR in Microbiology: From Diagnosis to Characterization
4. PCR Troubleshooting: The Essential Guide

Labels: PCR, PCR troubleshooting, real-time PCR