Computational protocol: Comparing EPA production and fatty acid profiles of three Phaeodactylum tricornutum strains under western Norwegian climate conditions

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Protocol publication

[…] For each strain, one sample from stock culture and four samples from different time points during the cultivation period were used for phylogenetic analyses. DNA was isolated from pelleted samples using the EZNA plant kit (stock cultures and samples from 25.04.16 and 17.08.16) or the MoBio PowerWater® DNA isolation kit (samples from 13.09.16 and 28.10.16) according to instructions from the manufacturers. PCR was performed using primers GF (5′-GGGATCCGTTTCCGTAGGTGAACCTGC-3′) and GR (5′-GGGATCCATATGCTTAAGTTCAGCGGGT-3′), which target ~ 820 bp of the variable region, ITS1-5.8S-ITS2, of the genome . The PCR mastermix contained (per 50 μL reaction); 25 μL Hotstart PCR mix, 2 μL 10% BSA, 1.2 μL 100% DMSO, 1 μL 10 μM primer GF, 1 μL 10 μM primer GR, 17.8 μL dH2O, and 2 μL template DNA. PCR conditions were: 15 min at 95 °C followed by 30 cycles of 1 min at 95 °C, 1 min at 55 °C and 1 min at 72 °C, and a final elongation at 72 °C for 7 min. Size of PCR products was confirmed using gel electrophoresis. PCR products were subsequently purified using ExoProStar™ (GE Healthcare, Chicago, Illinois, USA) according to the manufacturer's instructions, and prepared for sequencing using BigDye v.3.1 Kit (ThermoFisher Scientific, Watham, MA, USA) and the PCR GF/GR primer combination. Sanger sequencing was performed by the sequencing facility at the University of Bergen (http://www.uib.no/en/seqlab). Sequencing chromatograms were examined, and forward and reverse sequences were assembled and aligned using BioEdit (v.7.2.5) . A minimal evolution tree of these sequences and their closest relatives obtained from Genbank was constructed using Mega6 . […]

Pipeline specifications

Software tools BioEdit, MEGA
Application Phylogenetics
Organisms Danio rerio, Phaeodactylum tricornutum
Chemicals Eicosapentaenoic Acid