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PCR conditions were as follows: 2 min of initial denaturation at 94 °C, followed by a touchdown step of 30 s from 74 °C to 62 °C (due to the difference of the forward and reverse annealing primers), 35 cycles of 30 s at 94 °C and 30 s at 62 °C and a final extension step of 5 min at 72 °C.

PCR parameters were 95 °C for 5 min of initial denaturation, 95 °C for 30 s of denaturation, 59 °C for 2 min of annealing, 72 °C 4 min and 72 °C of 10 min for final elongation on Eppendorf Mastercycler personal, Germany.

After 10 min of initial denaturation at 95°C, the samples were subjected to the cycling parameters of 95°C for 15 s, 58°C for 30 s, and 72°C for 30 s (for 40 cycles) using a 7900 HT sequence detection system (Applied Biosystems, Foster City, CA).

The following thermal cycling conditions were used: 3 min of initial denaturation at 94 °C; 35 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 60 s, and elongation at 72 °C for 90 s; and a last step at 72 °C for 10 min.

Samples were obtained within 1.52 to 5.57 min (mean ± SEM  = 2.79±0.28 min) of initial disturbance.

Subjects in Study 2 were kept under identical conditions as those in Study 1. Baseline blood samples were collected from the brachial vein within 3 min of initial disturbance (2.28±0.12 min).

The PCR conditions were as follows: 5 min of initial enzyme activation at 95°C, 45 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s and elongation 72°C for 30 s. Subsequently, melting curves were obtained by measuring the drop in fluorescence when raising the temperature from 67 to 98°C with 5 observations per second.

The following cycling parameters were used: 3 min of initial denaturation at 94°C followed by 35 cycles of denaturation (30 s at 94°C), annealing (30 s at 55°C), and elongation (30 s at 72°C), with a final extension at 72°C for 6 min. The PCR results are shown in Figure S3.

After 5 min of initial denaturation at 95°C, reactions were carried out at the following conditions: denaturation, 95°C for 20 sec; annealing, 62°C for 20 sec; extension, 72°C for 30 sec; followed by a final elongation step of 72°C for 20 min. Triplicate samples were analyzed for each reaction and the mean values calculated.

After 2 min. of initial heat denaturing at 94°C, DNA was amplified by 40 cycles of denaturing at 94°C for 20 sec, annealing at 62°C for 20 sec and extension at 68°C for 20 s, followed by 5 min at 68°C.

Quantitative analyses of CHCs were done with a DB-1 fused silica column that was temperature-programmed from 150°C (1 min. of initial hold) at 5°C/min to 300°C.