Abstract
In the polymerase chain reaction (PCR) technique, DNA is amplified in vitro by a series of polymerization cycles consisting of three temperature-dependent steps: DNA denturation, primer-template annealing and DNA synthesis by a thermostable DNA polymerase. The purity and yield of the reaction products depend on several parameters. In this article we experimentally optimized the PCR thermal cycling parameters (primer/template annealing time and temperature, T ) and PCR reaction components (primera OPT combination and concentration, DNA polymerase and its concentration, concentration of polymerase buffer, template DNA concentration, dNTPs concentration and Mg ) for DNA amplification of Xanthomonas campestris 13551. It was found that 46°C suitable as T for maximum PCR amplification. Effect of annealinga OPT time on PCR amplification revealed that 30 seconds was ideal. Further, the effects of different concentrations of reaction components were seen on the amplification. It was found that 2mM dNTPs, 20μM primer concentrations of XcFwd2 and XcMMPE (0.356 kb) primer pair, X10 buffer and 2.5U of KOD Dash DNA polymerase and 10mM of Mg per 25 μl of reaction mixture yielded maximum product with no artifacts. Before applying any PCR based test, it is necessary to optimize the thermal cycling parameters and reaction conditions in individual laboratories.