Computational protocol: Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices

Similar protocols

Protocol publication

[…] For the construction of the 16S rRNA gene libraries, amplification was performed from total community DNA, obtained from each enrichment, by using the bacterial primer set 27f and 1100r. Only one library was assembled for each enrichment. Fifty microliter-reaction mixtures were made contained 50 ng of total DNA, 2 U of Taq DNA polymerase (Invitrogen), 0.2 mM of dNTP mix and 0.4 μM of each primer, in 1X Taq buffer. The PCR amplifications were performed using 10 cycles of 1 min at 94 °C, 30 s at 60 °C, decreasing 0.5 °C each cycle, and 3 min at 72 °C, followed by another 10 cycles of 1 min at 94 °C, 30 s at 56 °C and 3 min at 72 °C. Amplicons were pooled from five reactions (∼500 ng), purified using GFX™ PCR-DNA and gel band purification kit (GE Healthcare) and cloned using the pGEM-T cloning vector kit, according to the manufacturer's instructions (Promega, Madison, Wisc.). Insert-containing clones were submitted to ARDRA by digestion of M13 amplicons with the enzymes Hae III, Hha I and Msp I, independently, at 37 °C for 2.5 h. Clones representing distinct ribotypes were selected for DNA sequencing and phylogenetic affiliation.The 16S rRNA gene sequences were determined by direct amplification of the inserts from overnight grown clone cultures with M13 forward and reverse primers and sequencing with the DYEnamic ET Dye Terminator Cycle Sequencing Kit for the automated MegaBace 500 system (GE Healthcare) using the primers 10f, 1100r, 765f and 782r, according to the manufacturer's recommendations. Partial 16S rRNA gene sequences obtained from clones were assembled in a contiguous sequence using the phred/Phrap/CONSED program., Phylogenetic affiliation was achieved as described previously by Vasconcellos et al..The nucleotide sequences determined in this study were deposited at the Genbank database under the accession numbers: GenBank ID: JN998802 to JN998890. […]

Pipeline specifications

Software tools Phrap, Consed
Application Phylogenetics
Chemicals Polyurethanes