Cfx manager software ver 3.2.2

The bacterial DNA loads in the samples were assessed with qPCR,7 with the slightly modified procedure of Nadkarni et al.21 A reference curve relating the quantification cycle (C_q) to the bacterial DNA concentration in the samples was constructed with 6 10‐fold serial dilutions of Escherichia coli (ATCC 25922) DNA, starting from 1.97 × 10⁵ bacterial genome equivalents (GE) and diluted to 1.97 × 10⁰. The reference curve was incorporated into each qPCR experiment and used to determine the bacterial loads of the samples. The PCRs were performed in 20 μL containing 10 μL of SsoFast EvaGreen Supermix8 with 100 ng of DNA template. A 466‐bp fragment of the bacterial 16S rDNA was amplified on a CFX96 Touch instrument,8 under the following conditions: 98°C for 3 minutes and 40 cycles of 98°C for 5 seconds, 61°C for 1 minutes. Each reaction was run in triplicate. No‐template controls (NTCs) were included in each run. The data were analyzed with the Bio‐Rad CFX Manager software (ver. 3.2.2), supplied by the manufacturer. The PCR products were all of the expected length, were purified with the Wizard SV Gel and PCR Clean‐up System,9 in accordance with the manufacturer's recommended protocol, and were sequenced directly. The sequences were analyzed with the Basic Local Alignment Search Tool with the GenBank reference database to determine the source genera.