Computational protocol: Isolation of Uncultured Bacteria from Antarctica Using Long Incubation Periods and Low Nutritional Media

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[…] Genomic DNA was extracted using a PureLink Genomic DNA Mini Kit (Invitrogen, United States). 16S rRNA genes were amplified (initial denaturation step at 98°C for 5 min, followed by 30 cycles at 95°C for 30 s, 50°C for 30 s and 72°C for 2 min, followed by a final step at 72°C for 3 min) using the universal primers 27F and 1401R, purified using the GeneJet PCR purification kit (Thermo Scientific, United States), and sequenced using the BigDye Terminator kit (Applied Biosystems, United States), using the 27F, 1401R, and 518uF primers. Sequences were obtained by Sanger sequencing with an Abi Prism 3130xl Genetic Analyzer (Applied Biosystems, United States). Near the full sequence (1400 base pairs) of 16S rRNA was obtained. Sequences were deposited in Genbank under the access codes (KX990222.1 to KX990262.1). Phylogenetic trees were built using MEGA6 (), sequence alignments were performed with Muscle, and tree estimates were obtained using the maximum likelihood test (500 bootstraps). For sequence acquirement and analysis, EzTaxon () was used to obtain the top hits including type material sequences (described species). For environmental and cultured bacteria (non-type material sequences), Megablast (Genbank) top hits were used. In addition to EzTaxon and the NCBI database, our isolate’s sequences were also analyzed using the Silva database Project (). […]

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