Optimization of Polymerase Chain Reaction to Overcome Contamination of Deionized Water and Plumbing Premises By Pseudomonas spp. in Molecular Biology Laboratory
Abstract
The purpose of the current study is to introduce specific optimization steps to overcome non-specific binding of primers to contaminating DNA. The applied modifications provide applicable solutions especially if large number of primer aliquots were contaminated and the cost to replace them is high. Several steps were taken
to achieve complete mitigation of non-specific binding: reducing the concentration of both forward and reverse primers, reduction in the total number of PCR cycles from 35 to 25, increasing the annealing temperatures, doing filter sterilizations (0.2 µm Thermo Scientific polyethersulfone membrane) for the deionized water (DI) used in PCR and in certain cases reducing the extension time. The optimization steps carried in this work were successful in eliminating non-specific binding of primers to contaminating DNA found in primer aliquots.
Keywords: Plumbing Premises, Water Contamination, PCR, Pseudomonas, Deionized Water
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