Bacterial 16S rRNA Gene Cloning and Sequencing

Detail information for the cloning and transformation has been previously published (Kim et al. 2012 (link)). The primers used to amplify the 16S rRNA genes for bacteria were 27F and 1492R (Lane 1991 ). The amplification conditions for the PCR were 95°C for 5 min, followed by 30 cycles of 95°C for 45 sec, 55°C for 45 sec, and 72°C for 90 sec, with a final extension step for 5 min at 72°C. PCR products were purified with a QIAquick PCR purification kit (Qiagen, Valencia, CA) and ligated into a pUC118 HincII/BAP vector (Takara Bio Inc.), which was transformed into competent Escherichia coli DH5α cells (Invitrogen Corp., Carlsbad, CA) using heat shock. Plasmids from E. coli DH5α transformants were isolated using the PureLink Quick Plasmid Miniprep kit (Invitrogen Corp.). The 16S rRNA genes from the bacterial clones were sequenced using an Applied BioSystems model 3730xl automated DNA sequencing system (Foster City, CA).