The optimised temperatures for carrying out enzyme hydrolysis for the free and immobilised enzymes were found to be 50°C and 60°C, respectively.
Ablation volume was chosen as the characteristic to be optimised and temperature control was achieved via PID controller.
A hydroxyl-functionalised PMMA macroinitiator (HO-PMMA-Br) was prepared via ATRP at the optimised reaction temperature (60 °C) using 2-hydroxyethyl 2-bromoisobutyrate as the initiator.
With the exception of our optimised annealing temperature, the PCR reaction was carried out in duplicate using the Epitect MethyLight PCR Kit (QIAGEN) according to the manufacturer's instructions.
In the perfused leg, the optimised external temperature resulted in a small decrease in deep- Tmvl and in an increase in mid- and superficial- Tmvl (0.15 and 1.1 °C, respectively) compared to post-warm-up values.
The relative steepness of the gradient used to calculate the optimised annealing temperature is indicative of the thermal stability of the annealed template [ 14]: as demonstrated in Figure 3, there is an apparent 'trade-off' as the annealing temperature moves away from the optimal Tm.
Primer sequences and optimised annealing temperatures are given in Table 1.
The use of circulated liquid heating allowed the safe application of an optimised, higher heating temperature (43 °C) which resulted in a higher quadriceps Tsk (~38 °C; Table 1), thus minimising the temperature gradient between Tsk and Tmvl.
We hypothesised that the application of an optimised (higher) heating temperature will further reduce the previously observed drop in Tmvl over the course of the recovery period.
The pH of the reaction mixture, initial dye concentration, amount of enzyme and hydrogen peroxide concentration were optimised for ambient temperatures (30 ± 2 °C).
