Pcr purification kit

Rapid amplification of cDNA ends (RACE) special primers were designed by Primer Premier 5.0 according to sequence information of NR_026689 transcript. The 5′end of NR_026689 was obtained using the BD SMART^™ RACE cDNA Amplification Kit according to the manufacturer's instructions. Briefly, total RNA was isolated from lung tissues and reversed using the MMLV first Strand cDNA Synthesis Kit (BD Biosciences Clontech, CA, USA) and a PCR purification kit (Sangon, Shanghai, P.R. China). This was followed by the nested PCR with adaptor primer and gene-specific PCR primers. The primers used for nested PCR are shown in Supplementary Table S1.

Two clinical samples harboring wild-type (YMDD, ATG coding for methionine) and mutant (YIDD, ATT coding for isoleucine) DNA sequences confirmed by direct sequencing were used for plasmids construction. Target DNA was amplified with primers (KF, L1 and L2) listed in Table 1. PCR was performed in a 25-μl reaction mixture containing 12.5 µl of 2× HotStart Taq PCR Mastermix (Tiangen, China), 0.7 µl of each primer (10 µM), 9.1 µl of ddH₂O and 2.0 µl of DNA template. Thermal cycling conditions were as follows: initial denaturation at 94°C for 3 min, then 35 cycles with denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 45 s, and a final elongation step at 72°C for 10 min. PCR products were electrophoresed on a 2.0% agarose gel and purified via PCR purification kit (Sangon, China), then cloned into pMD 18-T vector (Takara, Japan) and transformed into E. coli DH5α competent cell (Takara, Japan). The positive plasmids were sequenced and then isolated using Tiangen mini plasmid isolation kit (Tiangen, China). Plasmids were quantified by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The corresponding copy number were calculated and 10-fold serially diluted from 1×10¹⁰ copies/μl to 1×10¹ copies/μl using Easy Dilution Buffer (Takara, Japan) to generate standard concentrations.

The detailed protocol of EMSA has been described in previous research [58 (link),59 (link)]. Briefly, approximately 300 bp promoter fragment of the candidate gene was amplified from A. caulinodans ORS571 genome and purified as a DNA probe using the PCR purification kit (Sangon Biotech, Shanghai, China). The DNA probes (50 ng) were mixed in a binding buffer containing different concentrations of RihR protein in a final volume of 20 µL. The binding buffer contained 50 mM Tris-HCl (pH 8.3), 0.25 M KCl, 2.5 mM dithiothreitol (DTT), 5 mM MgCl₂, 0.25 mg·mL⁻¹ bovine serum albumin, 0.05 mg·mL⁻¹ poly(dI-dC), 2.5 mM EDTA, 1% glycerol. The reaction mixtures were incubated for 20 min at room temperature and then electrophoresed on a 6% nondenaturing polyacrylamide gel in 0.5× Tris-borate-EDTA buffer at 150 V for 70 min. Gels were stained and photographed using GelRed (Sangon Biotech, Shanghai, China) and the molecular imager Gel Doc XR system (Bio Rad, Hercules, CA, USA).