( t.html Expand Long Templ...)PCR reaction components

t.html">Expand Long Template PCR System or Expand 20 kb PLUS PCR System. For most assays, the optimum amount of thermostable DNA polymerase (or a blend of polymerases) will be between 0.5 and 2.5 units/50 l reaction volume. Increased enzyme concentrations sometimes lead to decreased specificity. MgCl₂ concentration Mg2+ forms soluble complexes with dNTPs to produce the actual substrate that the polymerase recognizes. The concentration of free Mg2+ depends on the concentrations of compounds that bind the ion, including dNTP, free pyrophosphate (PPi) and EDTA. For best results, always determine the optimal Mg2+ concentration empirically. The optimal Mg2+ concentration may vary from approximately 1 mM to 5 mM. The most commonly used Mg2+ concentration is 1.5 mM (with dNTPs at a concentration of 200 M each). Mg2+ influences enzyme activity and increases the Tm of double-stranded DNA. Excess Mg2+ in the reaction can increase non-specific primer binding and increase the non-specific background of the reaction. Note: For easy optimization of MgCl₂, see the PCR Optimization Kit from Roche Applied Science (Cat. No. 1636138). Deoxynucleoside triphosphate (dNTP) concentration Always use balanced solutions of all four dNTPs to minimize polymerase error rate. Imbalanced dNTP mixtures will reduce Taq DNA Polymerase fidelity.
Note: For maximum convenience, a premixed, balanced mixture of dNTPs such as the '"/>

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