alias: 441In-515-806-2017
title: 16S of fish and parasite microbiomes
design: The 16S rDNA V4-V5 hypervariable region was amplified with E. coli 515f and 806r primers (with barcode on the forward primer) as specified by the Earth Microbiome Project 69-72. We used a 28 cycle PCR (5 cycle used on PCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94C for 3 minutes, followed by 28 cycles of 94C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongation step at 72C for 5 minutes was performed. After amplification, PCR products are checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Multiple samples are pooled together (e.g., 100 samples) in equal proportions based on their molecular weight and DNA concentrations. Pooled samples are purified using calibrated Ampure XP beads. Then the pooled and purified PCR product is used to prepare illumina DNA library. Sequencing was performed at MR DNA to obtain 250bp paired end reads (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturers guidelines.
identifiers.namespace: SUB8563803
identifiers.id: 441In-515-806-2017