From 2007.igem.org

PCR (Phusion Polymerase)

1. Set Up PCR Reaction:

     1 uL template DNA

     10 uL Phusion 5x HF Buffer

     1 uL 10mM dNTPs

     5 uL Primer Mix

     0.5 uL DMSO (optional)

     0.5 uL Phusion Polymerase

     Add H2O to total volume 50 uL

• Add the polymerase last and carefully.

• DMSO is used only for high GC content

• 5x GC Buffer may be used in replacement of HF Buffer for high GC content for Phusion kit

2. Run PCR machine

• Use 30 sec/kb for extension
• General cycle:

    1. Initial Denature	98ºC 		30 sec 

    2. Denaturation	        98 ºC	   	8 sec	

    3. Annealing		Annealing Temp	30 sec	

    4. Extension		72 ºC		30sec/kb

    5. Repeat steps 2-4 for an additional 29 cycles

    6. Final Extension	        72 ºC		10 min

    7. 			4 ºC 		forever

• Immediately place on ice or in 4 ºC after removing from PCR machine

*Cool Start Method for PCR (for more accurate amplification):

1. Keep all reagents on ice until use

2. Prepare the reaction mixture on ice

3. Set PCR machine ready to start with the designated program

4. Place tubes in PCR machine and start cycle immediately