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For less acute concerns, such as unilateral leg swelling developing over hours to days without severe pain or breathing difficulties, patients should contact their GP practice on the same day for assessment

denaturation at 95 C for 30 sec, annealing at 65 C for 30 sec, followed by the extension step at 68 C for 30 sec, repeated for 10 cycles, with the annealing temperature decreasing by 0.5 C in each cycle

Clifton et al

The peptide is now in a solution where it's far more vulnerable