U 2900 uv visible spectrophotometer

To determine the lipid oxidation of chicken breasts, the thiobarbituric Acid Reactive Substances (TBARS) values were analyzed using the method from Dias et al. [38 (link)] with few modifications. The sample (5 g) was homogenized (Digital Ultra-Turrax, Staufen, Germany) for 2 min with 20 mL of 5% trichloroacetic acid (TCA) and 0.5 mL of the artificial antioxidant 0.15% butylated hydroxytoluene (BHT, Sigma-Aldrich, Munich, Germany). After that, centrifugation of the homogenized samples was performed at 5000× g for 10 min. The obtained supernatants were filtered using Whatman No. 1 filter paper into 25-mL volumetric flasks and adjusted to 25 mL with 5% TCA. Two milliliters of the filtrate were mixed with two milliliters of 0.08% thiobarbituric acid (TBA) in glass tubes and were heated in a water bath at 95 °C for half an hour. Solutions in glass tubes were cooled down to room temperature, vortexed, and then absorbances were measured against a blank (mixing 2 mL of 5% TCA and 2 mL of 0.08% TBA) at 532 nm by U 2900 UV-visible spectrophotometer (Hitachi Ltd., Tokyo, Japan). TBARS values represented the mean of triplicate measurements for each sample (n = 3) and were reported as mg malondialdehyde/kg of meat sample [39 (link),40 (link)].

Oligonucleotides containing the G4 motif (or a d(GT)₈ insert) with single-stranded flanks (Figure 4, Table S4) were annealed in 20 mM Tris–HCl buffer (pH 7.3) containing 140 mM NaCl and 5 mM KCl (buffer A) to enable G4 formation. Direct annealing of an equimolar mixture of partially complementary oligonucleotide components (Figure 4, Table S4) in buffer A was used to prepare double-stranded DNA models. Absorbance versus temperature profiles of DNA samples (at ~3 µM concentration per oligonucleotide strand) were recorded in a 600-μL quartz microcuvette (Hellma Analytics, Germany) with an optical path length of 10 mm on a double-beam Hitachi U-2900 UV/visible spectrophotometer (Japan) equipped with a Hitachi thermoelectric controller. Changes in absorbance were monitored between 10 and 85 °C at 295 or 260 nm at a heating rate of 0.5 °C/min. T_m defined as the temperature of the mid-point, was estimated from a maximum/minimum value of the first derivative of the fitted curve for data smoothed with the Savitzky–Golay filter.

DNA oligonucleotides containing the G4 motifs were annealed in 20 mM Tris–HCl buffer, pH 7.3, containing 140 mM NaCl and 5 mM KCl (buffer A), or in 20 mM Tris–HCl buffer, pH 7.3, containing 140 mM NaCl and 0.5 mM KCl (buffer B) to promote G4 formation. One or two equivalents of PSM ligands were added to pre-annealed DNA probes; the mixtures were kept for 30 min at room temperature. The absorbance profiles of DNA samples as a function of temperature (at a concentration of ~3 µM per oligonucleotide strand) were recorded in a 600-μL quartz microcuvette (Hellma Analytics, Müllheim, Germany) with an optical path length of 10 mm on a double-beam Hitachi U-2900 UV/visible spectrophotometer (Tokyo, Japan) equipped with a Hitachi thermoelectric controller. Changes in absorbance were monitored between 20 and 85 °C at 295 nm at a heating rate of 1 °C/min. T_m, defined as the mid-point temperature, was estimated from the extremum value of the first derivative of the fitted curve for data smoothed with the Savitzky–Golay filter and indicated the M ± SD of three independent experiments.

UV absorption of DNA duplexes I–IV (0.6–1.0 µM) vs temperature was measured in a 300 µl quartz microcuvette (Hellma, Germany) with a 10 mm path length using the U-2900 UV/Visible Spectrophotometer (Hitachi, Japan) equipped with a thermoelectric temperature controller. The thermally induced unfolding transitions were monitored between 25 and 85°C at 260 nm with the heating rate of 0.5°C/min. Melting curves were obtained in integral form (at least 3 times for each duplex) and processed using GraphPad Prism 5 (GraphPad Software, Inc., USA). Hypochromic effect was calculated by the equation: . The duplex melting temperature (T_melt) and the ΔT values, which characterize the DNA melting cooperativity, were determined from the differential melting curves (Table 1). The mean SD was calculated.