Taqman fast universal master mix 2x no amperase ung

Concentrations of 50, 300 and 900 nM of each primer and 25, 50, 75, 100, 125, 150, 175, 200 and 225 nM of the probe were tested in order to select the optimal reaction conditions. The combination that gave the lowest threshold cycle (Ct) value and the highest final fluorescence was selected for the subsequent assays. The selected concentrations were 300 nM of SOFI_F primer, 900 nM of SOFI_R primer and 150 nM of SOFI_P probe.
Thus, each 20 µL reaction contained 10 µL of TaqMan Fast Universal Master Mix (2X), No AmpErase UNG (Applied Biosystems, Foster City, CA, USA), 1 µL of Primer SOFI_F (6 µM), 1 µL of Primer SOFI_R (18 µM), 1 µL of Probe SOFI_P (3 µM) and 100 ng of template DNA. Reactions were amplified in a 7500 fast real-time PCR System (Applied Biosystems, Foster City, CA, USA), with the fast ramp speed protocol: 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Samples were analyzed in triplicate, and Ct mean and standard deviation of each individual were registered.