alias: jcvi_cn_experiment_GS026_0.1_sanger_1.3KB
title: AB 3730xL Genetic Analyzer paired end sequencing; sanger sequencing of GS026 0.1µm filter fraction
design: sanger sequencing for use in comparative metagenomics of global surface oceans,                 GOS site GS026, 0.1µm filter fraction
library_construction_protocol: Environmental DNA (eDNA) was isolated from the impact filters and                 library construction was performed according to Rusch, et al. 2007. Briefly, shipboard frozen                 filters contained in the following storage buffer: 1X Tris-EDTA (TE) buffer (Invitrogen), 50mM                 EGTA and 50mM EDTA, were thawed to room temperature and aseptically cut for DNA extraction.                  Filter fragments were placed in 50ml conical tubes and lysis was performed as previously described.                  DNA was ultimately purified using a phenol/chlorofom extraction method followed by ethanol                  precipitation. For library construction using Sanger sequencing, eDNA was randomly sheared using                  nebulization and polished through BAL31 nuclease and T4 DNA polymerase reactions. Fragmented DNA                  was size selected via agarose gel electrophoresis. BstX1 adapters were ligated onto the inserts                  prior to ligation into the BstX1 linearized pBR322c2T plasmid vector used for shotgun library                  preparation. Libraries were then transformed and plated for isolation of colonies as sequencing                  templates.                                  Di-deoxy sequencing was used to generate paired-end sequence reads from plasmid templates.
identifiers.namespace: J CRAIG VENTER INSTITUTE
identifiers.id: jcvi_cn_experiment_GS026_0.1_sanger_1.3KB