Uv spectrophotometer 1650

Total phenolic content of all the extracts was evaluated with Folin-Ciocalteau method [32 ]. Samples containing polyphenols are reduced by the Folin-Ciocalteau reagent there by producing blue colored complex. The phenolic concentration of extracts was evaluated from a gallic acid calibration curve. To prepare a calibration curve, 0.5 mL aliquots of 12.5, 25, 50, 100, 200, and 400 μg/mL methanolic gallic acid solutions were mixed with 2.5 mL Folin-Ciocalteau reagent (diluted ten-fold) and 2.5 mL (75 g/L) sodium carbonate. After incubation at 25 °C for 30 min, the quantitative phenolic estimation was performed at 765 nm (UV Spectrophotometer 1650 Shimadzu, Japan). The total content of phenolic compounds was calculated in gallic acid equivalents (GAE) using the formula: A = (CXV)/m; where A is the total content of phenolic compounds, mg/g plant extract in GAE; C is the concentration of gallic acid established from the calibration curve, mg/ml; V is the volume of extract in ml and m is the weight of plant extracting.

For each SOMAmer reagent or duplex sample, 120–140 μl of the final solution was transferred to the micro-cuvette and read on a Shimadzu 1650 UV spectrophotometer at 260 nm. The cuvette holder was not moved during each individual melt, as this was found to generate baseline variability. Data were also collected while the sample was cooled at the same rate to ensure that there was no hysteresis in the transition temperature that would indicate that the heating/cooling rate was too fast. A buffer blank was subtracted from each data set. With each sample set, a natural DNA duplex with known composition and melting temperature was run as a positive control.
For the series A and series B duplexes, data were collected from 25–95°C, using a ramp rate of 0.5°C/min and a data collection rate of 0.5°C/min. For each SOMAmer reagent sample, data were collected from 5–95°C using a ramp rate of 0.5°C/min, and a data collection rate of 2 points/min. For the benzyl zipper duplexes, data were collected from 15–95°C, using a ramp rate of 0.5°C/min, and a data collection rate of 1 point/min. When samples were cooled below 25°C, a mild stream of argon gas was flowing in the sample chamber to prevent condensation.