Quant it picogreen double stranded dna assay

The DNA concentration of each PCR product was determined using a Quant-iT PicoGreen double-stranded DNA assay (Invitrogen, Germany). Following quantitation, the amplicons were subjected to emulsion PCR to generate amplicon libraries, as recommended by 454 Life Sciences. Amplicons of the samples from gut contents and surrounding surface sediments were subjected to pyrosequencing on the Roche 454 GS FLX Titanium platform at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.

The V3–V4 regions of the bacterial 16S rRNA gene were amplified from 50 ng of DNA extracts for each sample using primer sets 341F and 806R. Three replicates of 16S amplification were produced for the DNA sample. The triplicate PCR products were combined using an AxyPrep DNA Gel Extraction Kit (Axygen, Hangzhou, China) and a Quant-iT PicoGreen double-stranded DNA assay (Invitrogen, Carlsbad, CA, USA). The amplicons from reaction mixture were purified, pooled at an equimolar ratio, and subjected to emulsion PCR to set up the amplicon libraries. Subsequently, sequencing was performed using a Roche Genome Sequencer FLX Titanium platform. The generated DNA sequences were analyzed with the QIIME toolkit, version 1.9.1 [15 (link)].