The thermal cycling profile for the PCRs consisted of initial heating at 95°C for 15 min (hot-start polymerase activation), followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 90 sec, and ending with a 10 min extension step at 72°C.
Thermal cycling consisted of initial heating at 95°C for 5 min followed by 35 cycles of denaturation (1 min at 94°C), annealing (30 s at 57°C), and extension (1 min at 72°C).
If, for example, the initial heat of the expanding universe, or its total mass, or the strength of the force of gravity, or the mass of neutrinos, or the strength of the strong nuclear force, had been different by a small margin, there would have been no galaxies, no stars, no planets, and hence no life.
For thermocycling, the temperature steps of a TC-412 Thermal Cycler (Techne Incorporated, Burlington, NJ) were programmed for an initial heating at 85°C for 10 min, followed by 30 cycles at 85°C for 30 sec, 40°C for 30 sec, and 65°C for 1 min. The final extension was 65°C for 10 min as previously described [ 26- 28].
The PCR amplification was carried out for 35 cycles, where each cycle was 95°C for 1 minute, 55°C for 30 seconds, and 72°C for 30 seconds with an initial heat activation step of 95°C for 15 minutes and a final extension of 72°C for 10 minutes using TC 512 Thermocycler (Barworld Scientific, England).
The parameters used during the first test for the calculation of the initial heat exchange convective coefficients for the four surfaces are reported in Table 2.
PCR amplification was conducted in a PCR Thermal Cycler MP (TaKaRa Biomedicals, Ohtsu, Shiga, Japan) or an iCycler (Bio-Rad Laboratories) programmed for 5 min at 95°C initial heat activation and 30 cycles of 1 min at 95°C for denaturation, 1 min at appropriate temperatures (in Table 1) for annealing and 1.5 min at 72°C for extension, followed by 10 min at 72°C for final extension.
The first simulation was run at 310 K for 300 ps, using an initial heat time of 5 ps, with data collected every 0.01 picoseconds, with a time step of 0.002 ps, using NVT dynamics with a Berendsen thermostat [32].
For heat shock experiments, an initial heat shock was delivered and then fins were amputated 3 hr later.
