Takara ex taqtm dna polymerase

Conventional PCR was carried out using TaKaRa Ex TaqTM DNA polymerase (TaKaRa Bio Company, Kusatsu, Shiga, Japan) mixture with 10 ng DNA and 10 pmol each primer using a C1000 Thermal Cycler (BIO-RAD, California, USA). PCR conditions were as follows: pre-denaturation for 5 min at 95 °C, followed by 35 cycles of annealing and denaturation for 30 s at 95 °C, annealing for 20 s at 55–60 °C (depending on primer sequences), and extension 30 s at 72 °C, and final extension for 5 min at 72 °C. PCR products were amplified using target specific primers (CL_matK, CL_atpF, CL_ycf2, ZM_matK, ZM_atpF, and ZM_ycf2) and cloned using the RBC T&A Cloning Vector (Real Biotech Corporation, Taipei, Taiwan). Plasmid DNA was extracted from recombinant plasmids using the DokDo-Prep Plasmid Mini-Kit (ELPISBIOTECH, DaeJeon, South Korea) and sequenced by a commercial service (Macrogen, Seoul, Korea).

Total RNA from E. coli MG1656/pPintI2-1 (Table 1) was extracted and cDNA specific to the lacZ gene was synthetized by a reverse transcriptase (TaKaRa) using primers 30, 31, and 32 (Supplementary Table S1). After purification, cDNA was used as template for the 5′RACE experiment in accordance with the manufacturer’s recommendations (5′RACE System for Rapid Amplification of cDNA Ends, Invitrogen), and using the TaKaRa Ex Taq^TM DNA polymerase (TaKaRa Biotechnology). The purified PCR product was cloned in the pGEM^®-T Easy vector (Promega) in E. coli DH5aaa and nine clones were sequenced.