16S rRNA v34 amplicon sequencing of human stool
Purified DNA from stool samples was used to amplify the v34 region of the 16S rRNA gene by PCR. 50 ng of DNA was used as template with 1U of Taq Polymerase, 1x Taq buffer, 1.5 mM MgCl2, 0.4mg/mL BSA, 0.2 mM dNTPs, and 5 pmoles each of 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACNVGGGTWTCTAAT) Illumina adapted primers, as described by Bartram and colleagues (Bartram et al., 2011). The reaction was carried out at 94C for 5 minutes, 5 cycles of 94C for 30 seconds, 47C for 30 seconds, and 72C for 40seconds, followed by 25 cycles of 94C for 30 seconds, 50C for 30 seconds and 72C for 40 seconds, with a final extension of 72C for 10minutes. Resulting PCR products were visualized on a 1.5% agarose gel. Positive amplicons were normalized using the SequalPrep normalization kit (ThermoFisher #A1051001)