Phusion High-Fidelity Master Mix Protocol (E6040)

Protocol

1. Mix the following components in a sterile microfuge tube:
   
   DNA (3-25 ng):   1 μl
   Primer 1 (10 μM stock):   5 μl
   Primer 2 (10 μM stock):   5 μl
   Phusion™ High-Fidelity PCR Master Mix, 2X:   25 μl
   Sterile H₂O for a final volume of 50 μl:   14 μl
   --------------------------------
   Total volume:   50 μl
2. a. PCR cycling conditions for short amplicons (150-250 bp)

   Cycle step	Temp.	Time	Cycles
   Initial denaturation	98°C	30 sec	1
   Denaturation Annealing Extension	98°C 65°C 72°C	10 s 30 s 30 sec	6-18
   Final extension	72°C 4°C	5 min hold	1

   
   b. PCR cycling conditions for long amplicons (> 500 bp)

   Cycle step	Temp.	Time	Cycles
   Initial denaturation	98°C	30 sec	1
   Denaturation Annealing Extension	98°C 65°C 72°C	10 s 30 s 10-30 sec/1kb	6-18
   Final extension	72°C 4°C	5 min hold	1

3. Purify DNA sample on one column and elute in 50 μl.