alias: 20160317_sterivex_control_T_B
title: 16S rRNA gene sequencing of natural sea ice community
design: DNA extraction from Sterivex filters was performed using the PowerWater Sterivex DNA Isolation Kit (MO BIO Laboratories). Fluortex adsorbents were hung up using fishing line inside a 50 mL falcon tube and centrifuged for 30 min at 4700  g to recover the biofilm. Separating the pellet of biofilm cells from residual oil was facilitated by addition of 1 mL sterile filtered phosphate-buffered saline (PBS, pH 8). After removing the supernatant, the pellet was suspended in 0.9 mL of solution ST1B of the DNA isolation kit, transferred to a bead tube and further extracted according to the PowerWater protocol. For PCR amplicon sequencing, fragments covering the V3-V4 region of the bacterial 16S rRNA gene were PCR amplified using the primer pair Bac341F/Bac805R. The PCR products were supplied with Illumina adaptor overhang sequences in a second PCR. PCR purification, indexing with the Nextera XT Index Kit, library quantification, pooling and sequencing were performed following Illuminas protocol. Pooled libraries were sequenced on an Illumina MiSeq system using a 600 cycle MiSeq v3 Reagent Kit (Illumina) which produces two 300-bp long paired-end reads.
identifiers.namespace: SUB4176045
identifiers.id: 20160317_sterivex_control_T_B