alias: bacteria_and_archaea
title: 16S rRNA gene amplicon sequencing of Southern Ocean microorganisms exposed to acidification
design: Methods of MiSeq, taken from MR DNA (www.mrdnalab.com, Shallowater, TX, USA). The 16S rRNA gene V4 variable region PCR primers 515/806 (OR whichever PRIMER set the investigator has SELECTED) with barcode on the forward primer  were used in a 28 cycle PCR  (5 cycle used on PCR products) using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94 C for 3 minutes, followed by 28 cycles of 94 C for 30 seconds, 53 C for 40 seconds and 72 C for 1 minute, after which a final elongation step at 72 C 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 (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer s guidelines. MR DNA joins the raw reads and reorientates reads into a uniform direction
identifiers.namespace: SUB3340652
identifiers.id: bacteria_and_archaea