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==Magnesium concentration==
Magnesium is required as a co-factor for thermostable DNA polymerase.It acts as a catalyst in the reaction without being consumed.Taq polymerase is a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction.<ref name="Markoulatos2002">{{cite journal |vauthors=Markoulatos P, Siafakas N, Moncany M |title=Multiplex polymerase chain reaction: a practical approach |journal=J. Clin. Lab. Anal. |volume=16 |issue=1 |pages=47–51 |year=2002 |pmid=11835531|doi=10.1002/jcla.2058}}</ref> Some of the components of the reaction mixture such as template concentration, dNTPs and the presence of [[chelating agents]] ([[EDTA]]) or [[proteins]] can reduce the amount of free magnesium present thus reducing the activity of the enzyme.<ref name="Promega">{{cite journal|title=Nucleic acid amplification protocols and guidelines|url=http://www.promega.com/paguide/chap1.htm}}</ref> Primers which bind to incorrect template sites are stabilized in the presence of excessive magnesium concentrations and so results in decreased specificity of the reaction. Excessive magnesium concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA during PCR reducing the product yield.<ref name="Markoulatos2002"/><ref name="Promega"/> Inadequate thawing of MgCl<sub>2</sub> may result in the formation of concentration gradients within the [[magnesium chloride]] solution supplied with the DNA polymerase and also contributes to many failed experiments .<ref name="Promega"/>
== Size and other limitations ==
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