Magnesium ConcentrationMagnesium 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.
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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.
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