Computational protocol: Thermostable and Alkalistable Xylanases Produced by the Thermophilic Bacterium Anoxybacillus flavithermus TWXYL3

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Protocol publication

[…] Total genomic DNA was isolated from mid-log phase cultures using the DNeasy Tissue Extraction Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. PCR-mediated amplification of the 16S rRNA gene was performed using primers 8F and 1492R [] along with Platinum Taq polymerase (Invitrogen Corp., Carlsbad, CA). Sequencing of the PCR products was performed using 8F, 338F, 338R, 907F, 907R, and 1492R []. All DNA sequences were determined by the Idaho State University (ISU) Molecular Research Core Facility. The 16S sequences obtained were aligned using sequences obtained from the nonredundant NCBI database (http://www.ncbi.nlm.nih.gov) and the CLUSTALW program found in the BioEdit program (version 7.0.5.3, Department of Microbiology, North Carolina State University, Raleigh, NC). Sequences from the full-length 16S rRNA genes from multiple closely related characterized organisms resulting from BLAST, along with multiple distantly related organisms, were BLAST-searched and used in the alignment to establish relationships among TWXYL3 and phylogenetically related isolates. Eighteen sequences were used in the alignment and employed in maximum-likelihood analysis utilizing the PAUPBOOT package (Luobin Yang, Idaho State University) with 100 bootstraps. Escherichia coli strain KCTC 2441 (EU014689.1) was used to root the tree. […]

Pipeline specifications

Software tools Clustal W, BioEdit
Application Phylogenetics
Organisms Anoxybacillus flavithermus
Chemicals Carbohydrates