Nucleospin rna plant

We isolated the total RNA from the inflorescence meristem of grape and Arabidopsis plants. Young grape inflorescences of 3–8 mm in the longitudinal direction of each cultivar were used. The inflorescence meristems of 20-day-old Arabidopsis plants before the appearance of an inflorescence were used. After freezing these samples with liquid nitrogen, the samples were homogenized with an SK mill (SK-200) (Tokken, Kashiwa, Japan). According to the manufacturer’s instructions, total RNA was isolated from these homogenized samples using Nucleospin RNA plant (Takara, Otsu, Japan).

The Escherichia coli strains used in this study were DH5α for plasmid propagation, and BL21 (DE3) star pRARE and Rossetta2 (DE3) pLysS (Invitrogen, Carlsbad, CA, USA) for protein expression. C. thermophilum was obtained from NBRC (Biological Resource Center, National Institute of Technology and Evaluation, Tokyo, Japan). The protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard. Total RNA of C. thermophilum was obtained using the NucleoSpin RNA Plant (Takara Bio, Shiga, Japan), and cDNA was synthesized by the cDNA Synthesis Kit (Takara Bio). KOD-Plus-Neo DNA polymerase used for gene amplification and restriction endonucleases were obtained from TOYOBO (Osaka, Japan) and New England Biolabs Japan (Tokyo, Japan), respectively. Nucleotides and other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) or Sigma-Aldrich Japan (Tokyo, Japan). Actin was extracted from an acetone-dried powder made from chicken white breast meat, and was purified by ultracentrifugation, as previously described for rabbits [28 (link)]. Tubulin was purified from porcine brain with polymerization and depolymerization cycling [29 (link)]. CtCCT was expressed and purified as described previously [19 (link)].

Total RNA was extracted from seedlings using NucleoSpin® RNA Plant (Takara Bio.) which includes DNase I treatment. Reverse transcription was performed using a PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio.), and PCR was performed using KOD One® PCR Master Mix (TOYOBO) and the RT product as a template. Thirty-five cycles of PCR were performed for detection of RTs and 30 cycles for detection of the first genes in 20 μl reaction volume. Primers used for PCR are listed in Supplementary Table 2. PCR products were loaded onto a 1.5% agarose gel and electrophoresed in TBE buffer followed by detection under ultra-violet (UV) light using GelRed stain (Nacalai Tesque, Japan). As size markers, Gene Ladder 100 (Nippon Gene) or GD 1Kb plus DNA ladder RTU (GeneDireX) were used.