Cfx96 touch real time thermal cycler

All commercial thermal-block-based PCR runs were performed in a CFX96 Touch real-time thermal cycler (Bio-Rad) or SimpliAmp thermal cycler (Life Technologies). Polypropylene plastic tubes and glass capillary tubes were also used to demonstrate the TTC’s flexibility in terms of accepting various tubes to deliver fast heat transfer in water. While there is no heated lid in our design, and we noticed that some evaporation and condensation occurred, we found that the quality of the PCR amplification was not significantly affected when checked by gel electrophoresis. In this manuscript, the template concentrations used in PCR reactions were purposely chosen to be very realistic, with most of them giving a Cq of 25 to 34, which allows us to access and demonstrate the efficiency of the TTC. Table 1 summarizes the targets, primer sequences, product sizes, and components of the PCR reactions. The individual PCR and RT-PCR run protocols will be described in the corresponding data sections. Stock primer concentrations were 10 μM and were diluted to a final concentration of 500 nM after the master mixes were prepared. Unless specified otherwise, reaction volumes for the commercial cycler and TTC were 20 μL when polypropylene tubes were used. The sample volume for glass capillary tubes was 17 μL.