16S bacteria amplicon seq of In situ Incubation of passive sampler in groundwater of anoxic well, 42 weeks, replicate 2
PCR amplification of the bacterial V3-V4 region of the 16S rRNA was performed in a 25 L reaction. Illumina sequencing adapters AATGATACGGCGACCGAGATCTACAC and CAAGCAGAAGACGGCATACGAGAT were attached at the 5 ends of the forward and reverse primers, respectively. PCR was conducted using primer combinations Bakt_0341F/Bakt_0785R (Krger et al., 2021). Cycling conditions included initial denaturation at 95 C for 15 min, 25-28 cycles at 94 C for 45 s, 55 C for 45 s, 72 C for 45 s, and final elongation at 72 C for 10 min. Amplicon sequencing was performed inhouse using a MiSeq Illumina platform (Illumina, Eindhoven, The Netherlands) with v3 chemistry.