Kzo9i

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.