Taq dna polymerase buffer

PCR-RFLP based method was used to detect polymorphism in IL-18 gene. Three SNPs, rs1946519 (−656 G/T) and rs187238 (−137 G/C) of promoter region and rs549908 (+105 A/C) of codon region, were selected for the study. For each polymorphism, a specific PCR-RFLP was done using method described previously [13 (link)] with few modifications (Table 2). PCR was performed in 25 μL reaction mixture using 100 ng genomic DNA with 1.5 U Taq DNA polymerase (New England Bio labs, UK) supplemented with 10X Taq DNA polymerase buffer, 1.5 mM MgCl₂, 25 μM dNTPs, and 10 pM of forward and reverse primers. Each amplified product was digested by specific restriction enzymes (Fermentas) and electrophoresed on a 3% (w/v) ethidium bromide (Merck, Mumbai) stained agarose gel (Sigma-Aldrich, St. Louis, USA) and visualised on UV transilluminator.

RNA electrophoretic mobility-shift assay (REMSA) was performed as described previously (Lin and Xu, 2012 (link)). Briefly, to prepare the template, equimolar oligonucleotides were mixed, heat-denatured, and annealed in Taq DNA polymerase buffer (NEB). Next, the template (0.5 μM final concentration) was used for in vitro RNA transcription by T7 RNA polymerase (NEB) following the manufacturer’s instructions. The templates were digested with RNase-free DNase I (TaKaRa), after which RNA probes were purified with the RNeasy Mini Kit (Qiagen) and then incubated with WTG1 protein at 30 °C for 30 min in a 20 μl system containing 20 mM Tris-acetate (pH 7.9), 50 mM potassium acetate, 10 mM magnesium, 2.5 mM dithiothreitol, 500 μg/ml BSA, and 40 units of RNase inhibitor. To prevent non-specific binding, 500 μg/ml BSA was added to the reaction system. RNA–protein complexes were resolved on a 1.5% agarose gel and detected by GelRed staining using the methods described by Lin and Xu (2012) (link).

Transgenic maize (Zea mays Hi II hybrid A 188 and B73 background) expressing the glob::Enz cassette were produced by the Iowa State University Plant Transformation Facility (www.biotech.iastate.edu) using Agrobacterium-mediated transformation protocol [53 ]. Initial plant material was provided by the facility. Plantlets from ten putative transgenic lines were obtained after tissue-culture selection on media containing the selectable agent, bialophos. Each transgenic line was confirmed by PCR to be containing the glob::Enz cassette by genomic PCR using primers specific to the cassette (Enz-For 5′-GTTGGCAGATCTTAACGCTCT-3′, Enz-Rev 5′-CTTCCCATTCAGCCCTACCTC-3′ producing an expected amplicon of 743 bp). Standard PCR conditions were used in the PCR screening: 50 ng genomic DNA, 1X Taq DNA polymerase buffer, 2U Taq polymerase (New England Biolabs), 250 μM of each dNTP and 200 nM of each primer with PCR conditions of an initial denaturation (94 °C, 4 min) and 45 amplification cycles (94 °C, 30 s; 55 °C, 30 s; 72 °C 60 s) followed by a final elongation step (72 °C, 7 m). Transgenic lines were grown to the T₃ generation by repetitive self-pollination and ensured stable transmission of the Enz cassette by performing Enz-specific PCR reaction screening on progeny each generation.