Comparative Evaluation of DNA Polymerase Reagents

Four different DNA Polymerase reagents were tested as part of this study: GoTaq Green Master Mix (Promega, Madison, WI, USA), Phusion Plus PCR Master Mix (Thermo Scientific, Waltham, MA, USA), AmpliTaq Gold Master Mix (Applied Biosystems, Waltham, MA, USA), and Hemo KlenTaq (New England BioLabs, Ipswitch, MA, USA). The gene chosen for amplification was the 16s rRNA gene and the primers used have been published previously [26 (link)]. Following PCR, the amplified DNA was run on a 1% agarose gel, stained with ethidium bromide, and imaged using a Bio-Rad Gel Doc EZ Imager (Bio-Rad, Hercules, CA, USA). Gel images are shown in Supplementary File S2. All reagents were used following protocols described by their respective manufacturers and run in a Bio-Rad T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). DNA extracted through the phenol–chloroform method yielded higher concentrations than the other two extraction methods; therefore, 0.5 µL of DNA from each sample was used for PCR. The exact protocol details and thermocycler conditions for each reagent are as follows: