Fluorescence versus temperature profiles of DNA duplexes were measured by a Bio-Rad CFX96 Touch Real-Time PCR. Each duplex was formulated at 6 different concentrations (1, 5, 10, 50, 100, 300 uM) in the buffer with 1X EvaGreen fluorescence dye, and each concentration was replicated 3 times to reduce experimental error. The solution was annealed and then subjected to high resolution melt. Melt curves of duplexes were monitored from 5 ℃ to 90 ℃, and fluorescence data were recorded every 0.2 ℃. Almost all oligonucleotides were in duplex states at low temperatures, EvaGreen specifically intercalated duplexes results in high fluorescence in the buffer.