Computational protocol: Bioprospecting North Atlantic microalgae with fast growth and high polyunsaturated fatty acid (PUFA) content for microalgae-based technologies

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Protocol publication

[…] Molecular methods were used to give taxonomic information of the isolates. DNA was isolated from the pellet of 10 mL algal culture (harvested at 1663g for 6 min) using E.Z.N.A. SP Plant Kit (Omega Bio-tek, Inc., Norcross, GA, USA). Microalgal DNA was amplified by PCR using the HotStarTaq DNA Polymerase Kit (QIAGEN, Valencia, CA, USA), according to the manufacturer's instructions. Amplification of a region of the 28S ribosomal RNA (rRNA) gene (for the large subunit [LSU] of eukaryotic cytoplasmic ribosomes) was performed using the primers D1R-F and D3B-R . The reaction conditions were as follows: An initial activation of the enzyme at 95 °C for 15 min, followed by 30 cycles of denaturation (94 °C, 1 min), annealing (56 °C, 1 min) and extension (72 °C, 1 min), and a final extension at 72 °C for 10 min. Before sequencing, the PCR product was purified with GenElute™ PCR Clean-Up Kit (Sigma-Aldrich, St. Louis, MO, USA) and quantified with Qubit ® dsDNA BR Assay Kit and Qubit® 2.0 (Invitrogen, Eugene, Oregon, USA). Bi-directional sequencing of the PCR products was performed using the PCR forward and reverse primers with the BigDye v.3.1 Kit (ThermoFisher Scientific, Watham, MA, USA) at the sequencing facility at the University of Bergen (http://www.uib.no/en/seqlab). Sequences were edited and aligned manually in BioEdit , and Blastn was used to search for similarities with previously published diatoms in GenBank (www.ncbi.nlm.nih.gov/blast/Blast/cgi). […]

Pipeline specifications

Software tools BioEdit, BLASTN
Application Phylogenetics
Chemicals Fatty Acids, Fatty Acids, Unsaturated, Eicosapentaenoic Acid