High pure dna purification kit

Digestion with AluI and SmaI restriction enzymes, ligation with the T₄DNA ligase, and dephosphorylation using calf intestinal alkaline phosphatase were performed according to the supplier’s recommendations (New England Biolabs, Leiden, The Netherlands). The R-IVET library was constructed in the STUL5003 strain. The genomic DNA of S. thermophilus LMD-9 was partially digested with AluI, and restriction fragments were ligated with the pULNcreB plasmid, which was previously linearized by SmaI and dephosphorylated. DNA digestions were checked by agarose gel electrophoresis and were purified using the High Pure DNA purification Kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s recommendations. Chemically competent cells of E. coli TOP10 were transformed with the ligated plasmids. All resulting clones were pooled and their plasmid DNAs extracted using a Miniprep Kit (Fermentas, Villebon sur Yvette, France). Plasmid DNAs were then introduced by natural transformation into S. thermophilus STUL5003. The resulting S. thermophilus clones were selected on LM17 supplemented with streptomycin, pooled, resuspended into LM17 supplemented with 11.6% glycerol, and stored in aliquots at −80 °C to constitute the S. thermophilus R-IVET genomic library.

E. coli strains DH5α and BL21 (DE3), and pET26b (+) vector were purchased from Invitrogen (USA). Restriction enzymes were provided by Fermentas (USA). PCR product extraction and high pure plasmid purification kits were purchased from Bioneer (Korea). High pure DNA purification kit was purchased from Roche (Germany). T4 DNA Ligase, isopropyl β-D-1-thiogalactopyranoside, and PVDF membranes were purchased from Thermo Fisher Scientific (Germany). Casein, azo-Casein, 1 anilino naphthalene-8-sulfonate (ANS), and anti-polyHistidine peroxidase antibody were purchased from Sigma (USA). All other chemicals were obtained from Merck (Germany).