Amplicon sequencing of the methanogen-specific mcrA gene to study microbial DMS degradation in river sediments
mcrIRD primer set (forward: 5-TWYGACCARATMTGGYT-3, reverse: 5-ACRTTCATBGCRTARTT-3). Two-step PCR: 1-PCR: 1 L DNA, 1 L/primer (10 M), 25 L 2x MyTaq HS Red Mix, and 22 L ultra-pure water. Conditions: 95 oC for 5 min, 39 cycles of 95 oC for 1 min, 51 oC for 1 min, 72 oC for 1 min. 72 oC for 5 min. 2-PCR: 2 L of the clean 1-PCR product, 0.5 L/primer containing the overhang adapters (10 M), 12.5 L 2x MyTaq HS Red Mix, and 9.5 L ultra-pure water. Conditions: 95 oC for 3 min. 15 cycles of 95 oC for 20 sec, 55 oC for 15 sec, 72 oC for 15 sec. 72 oC for 5 min. Further amplification added dual indices and Illumina sequencing adapters.