1. Components Of A Typical PCR Reaction Mix PCR Reaction Buffer (usually supplied at 10X) 100mM Tris-HCl - Essentially a pH Buffer. Maintains appropriate molecular working environment for enzyme(s) involved. Note: pH of reaction can affect fidelity. For example, with respect to Taq polymerase, fidelity is varies inversely with pH. 500 mM KCl - Assists in primer/template annealing. High concentrations may result in inappropriate products due to stabilized mispriming events. 15 mM MgCl 2 - Required for the activity of Taq polymerase. It is the free Mg 2+ that is important. Free Mg 2+ concentration is inversely related to the dNTP concentration of the solution. Free Mg 2+ concentration also affects Taq polymerase fidelity, as fidelity has been demonstrated to vary inversely with free Mg 2+ concentration.

2. Components Of A Typical PCR Reaction Mix (Continued) 50 - 200 µM dNTPs Remember dNTP concentration can affect efficiency as well as fidelity of Taq polymerase. Modified dNTPs And Their Uses 7-deaza-dGTP - reduction of secondary structure in G-rich DNA dUTP - used for contamination prevention by allowing for enzymatic destruction of PCR products from previous amplficiations that may be present in a reaction mix. Radiolabeled and nonradioactively labeled dNTPs - detection of PCR product Dideoxy and flurorescently labeled dideoxy dNTPs - Chain termination/detection in DNA sequencing dPTP and 8-oxo-dGTP - introduction of random mutations in PCR products (Error Prone PCR)

3. Components Of A Typical PCR Reaction Mix (continued) Primers - Oligonucleotides which direct the synthesis of DNA by providing a double stranded region with a free 3’ OH to which dNTPs can be added. Characteristics Of PCR Primers: Length: 20-30 nucleotidesAnnealing Temp: Approximately 50 o C* Base Composition: Equal Amounts Of Each Base Possess correct directionality Lack of repetitive sequences or runs of the same nucleotide Lack of ability to form secondary structure Lack of ability to form primer dimers Lack runs of 3 or more G’s or C’s at the 3’ end Note: It is essential that the 3’ end of the primer be complementary to the template. This is not true of the 5’ end. Also, when calculating primer annealing temperature, be sure to include only the region that anneals to the template. * Varies with application

4. Determining Primer Annealing Temperature The temperature at which 50% of the primer molecules in solution are annealed, called melting temperature or T m, is used as an estimate of the primer annealing temperature of a PCR reaction. For primers up to about 20 nucleotides in length, or for a “quick & dirty” estimate, the following equation is useful: T m = 4(number of G + C) + 2(number of A + T) For primers between 15 and 70 nucleotides in length, or for greater accuracy, use the following equation: T m = 81.5 + 16.6[log(I)] + 0.41(%G + C) - (600/N), where I is the molar concentration of monovalent cations and N is the length of the primer. In some cases, the optimized annealing temperature (T p ) is used: T p = 22 + 1.46[2(number of G + C) + (number of A + T)] Important: You should consider only those primer regions that actually anneal to your template when performing these calculations.