Deconstructing microbial community substrate utilization: Differential incorporation of dissolved organic carbon types by diverse populations of Sargasso Sea bacterioplankton
The polymerase chain reaction (PCR) was used to amplify the V1-V2 hypervariable regions of the 16S small ribosomal RNA subunit gene from each sample. PCR reactions used 10 µL template (except Raw Whole: 2 µL) in a 25 µL reaction mixture containing 1.5 Unit PerfectTaq Polymerase (5Prime) in proprietary buffer, 50 nM each dNTP, 2.5 mmol L-1 additional MgCl2 (4 mmol L-1  final), 5% Acetamide, and 200 nM each forward and reverse primers. Primers used were forward primer 8f (5’-FAM-AGRGTTYGATYMTGGCTCAG) and reverse primer 519r (GWATTACCGCGGCKGCTG) and in library construction we used forward primer 8f (with 5’ Roche FLX Amplicon Adapter B  GCCTTGCCAGCCCGCTCAG followed by TC linker) and reverse primer 338r (TGCWGCCWCCCGTAGGWGT with 5’ Roche FLX Amplicon Adapter A and integrated barcode GCCTCCCTCGCGCCATCAGxxxxxxxxCA where x’s refer to oligonucleotide ‘barcodes’)(Hamady et al. 2008). PCR reaction conditions in library prep were 270s 94°C hotstart, 35 cycles of 30s 94°C, 60s 57°C, 120s 72°C, 600s 72°C extension and 4°C storage.