After an initial activation at 50°C for 2 min and a preincubation step at 95°C for 10 min, reaction mixtures were subjected to 40 cycles of denaturation at 95°C for 15 sec, annealing at 56°C for 30 sec and extension at 72°C for 35 sec (ABI 7500).
The increasing effect in cytotoxic acivity of DOX was conserved even when the drug was added 10 min after the preincubation step with NADPH and CPR (data not presented).
PCR conditions consisted of 10 min of a preincubation step at 95°C followed by 45 cycles of denaturation (95°C, 10 sec), annealing (60°C, 30 sec), and extension (72°C, 1 sec), followed by a final cooling step of 30 sec at 40°C.
The data obtained also evidenced that it was (they were) (a) relatively long-lived specie(s) because the increasing effect in cytotoxic activity of DOX was conserved even when the drug was added 10 min after the preincubation step with NADPH and CPR.
For autoradiography, a preincubation step of 15 min in Tris buffer (50 mM, pH 7.4) was added to remove exogenous TSPO ligand.
The thermal cycle consisted of a preincubation step of 2 min at 94°C, 30 cycles at 94°C for 30 s, a specific annealing temperature of 60°C for 10 s and an extension phase at 72°C for 20 s.
The thermal cycle consisted of a preincubation step of 2 min at 94°C for complete denaturation of the DNA followed by 31 cycles (27 cycles for exon 7) at 94°C for 30 s, a specific annealing temperature of 60°C (exons 5, 7 and 8) or 65°C (exon 6) for 10 s and an extension phase at 72°C for 10 s.
Once transferred, the solution was preincubated for 20 min. During the actual screen this preincubation step facilitates diffusion throughout the well and also allows for any covalent or slow binding compounds to interact with the enzyme.
The amplification program consisted of a preincubation step (95°C for 5 min), followed by 35 cycles consisting of a denaturation step (95°C for 30 s), an annealing step (56°C for 30 s) and an extension step (72°C for 30 s), and then one final extension step (72°C for 7 min).
The PCR conditions consisted of an uracil-N-glycosylase preincubation step at 50°C for 2 min, followed by a denaturation step at 95°C for 10 min, and 40 cycles of amplification (denaturation for 15 sec at 95°C, annealing and elongation for 1 min at 60°C).
