Culturing_454_16S_GE82GC3
The V2 region of bacterial 16S rRNA genes was subjected to PCR amplification. PCR reactions were carried out in triplicate using 2.5x Master Mix (5 Prime), forward primer (FLX-8F; 5'-GCCTTGCCAGCCCGCTCAGTCAGAGTTTGATCCTGGCTCAG-3Õ), barcoded reverse primer (FLX-BC-338R; 5'-GCCTCCCTCGCGCCATCAGNNNNNNNNNNNNCATGCTGCCTCCCGTAGGAGT-3Õ; barcode sequence is shown as NÕs), and 50 ng input DNA. Reactions were incubated for 2 min at 95 degrees C, followed by 30 cycles of 20 sec at 95 degrees C, 20 sec at 52 degrees C, and 1 min at 65 degrees C. Triplicate reactions were pooled, inspected by gel electrophoresis, and purified on AMPure beads (Agencourt Biosciences). For each barcoded primer, negative control reactions lacking input DNA were conducted in parallel. 