MCR RR 2010 with DCNS
Coral and macroalgal exudates vary in neutral sugar composition and differentially enrich bacterioplankton populations in a tropical reef ecosystem
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 2 *L template in a 25 *L 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 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, 45s 57*C, 60s 72*C, 600s 72*C extension and 4*C storage.
16S rRNA amplicon library Nelson 2
Marine Science Institute
MCR RR 2010 with DCNS