Team:Freiburg/Protocols/Fusion PCR
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Fusion PCR
PCR mix I
PCR run I
Repeat steps 2 till 4 for 5 times.
** Calculated annealing temperature of the whole owerlap (OligoCalc, basic). If the temperature of step 3 is close to or higher than 72 °C, a touchdown PCR should be performed: The temperature of step 3 will be reduced for 0.5 °C every cycle, starting at 72 °C.
PCR run II
Add 2.5 μl of each primer that anneal at the ends of the new construct.
Repeat steps 2 till 4 for 18 times.