Answer to Question #113146 in General Chemistry for Vince Porsgaard

Question

Magnesium chloride:
MgCl2 is a cofactor for the enzyme Taq polymerase. Higher concentrations of MgCl2 increase activity of the enzyme, but also reduces specificity. To determine the ideal concentration of MgCl2 in your experiment, you set up a series of reactions varying its concentration. Determine the amount of MgCl2 you need to add to each PCR tube in each case, if the stock solution is at 25mM:
a) 1.5 mM
b) 2.5 mM
c) 3.5 mM

Answer 1

The 3'-OH of the growing chain contains a lone pair of electron which is used for the formation of phosphodiester bond during the process of polymerization by Taq Polymerase (or any polymerase). This lone pair of electrons can do nucleophilic attack on the phosphate atom of alpha-phosphate, releasing the beta and gamma phosphate. But the incoming nucleotide has 4 negative charges (dNTP4-) and due to the presence of these many negative charges the nucleophilic attack is retarded. Now, Mg2+ comes to rescue by chelating extra negative charges of the incoming dNTP, facilitating the nucleophilic attack and bond formation, hence, polymerization.

This complete process in brief is generally referred as increment in polymerase activity as the polymerase (containing asp residues in their active sites to house the Mg2+ ions) is directly benefited.

In short, you increase Mg2+ in PCR, you get strong band but chances of non-specific amplification also rises. Reduce Mg2+, you get rid off non specificity. 1.5mM to 3.5mM of MgCl2 in final reaction mixture is the best range to work with.

so best answer is 2.5mM