PCR with Phusion-HF DNA Polymerase (NEB):

Using the guidelines of the provided Phusion® High-Fidelity DNA Polymerase (M0530) protocol.

1. Reaction Setup:
   
   10 µl 5X Phusion HF Buffer
   5 µl dNTPS [2 mM]
   2.5 µl Forward Primer [10 µM]
   2.5 µl Reverse Primer [10 µM]
   1 µl Template DNA
   0.5 µl Phusion HF DNA Polymerase
   28.5 µl MilliQ Water
   

   ---

   50 µl Total Reaction




2. PCR Program:
   

   Step	Temperature	Time
   Initial Denaturation	98 °C	30 seconds
   35 Cycles	98 °C	10 seconds
   	45 °C – 72 °C	30 seconds
   	72 °C	30 seconds / 1 kb
   Final Extension	72 °C	5 minutes
   Hold	4 °C	forever