Agarase agarace

Citrobacter sp. 36-4CPA was isolated from hydrocarbon-contaminated soil samples. The isolate was identified by 16S rRNA gene phylogenetic analysis as a member of the polyphyletic genus Citrobacter. Genomic DNA was isolated from bacterial culture, as previously described (Boulygina et al., 2002 ). The 16S rRNA partial gene (1480 bp) was amplified by using universal primers 27f and 1492r (Edwards et al., 1989 (link)). PCR products were purified by LMP (low melting point) agarose and agarase AgarACE™ (Promega). The partial 16S rRNA gene was sequenced using universal primers 27f and 1492r with a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on a 3730 DNA Analyser (Applied Biosystems) according to the manufacturer’s instructions.

Plasmid DNA was isolated by Birnboim–Doly alkaline lysis method (Birnboim and Doly, 1979 (link)). Purified through LMP (low melting point) agarose and agarase AgarACE™ (Promega), plasmid DNA was analysed by 1% agarose gel electrophoresis. The plasmid was digested with the restriction endonucleases Kzo9I, RsaI and AluI (Promega). Later, Kzo9I, RsaI and AluI fragments were cloned into pGEM-3Zf(+) (Promega). The recombinant plasmid DNA was then transformed into Escherichia coli DH5α and isolated with a Wizard MaxiPrep kit (Promega). DNA sequencing was performed using universal M13 primers with a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on a 3730 DNA Analyser (Applied Biosystems) according to the manufacturer’s instructions.