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	*Immediately place on ice or in 4 ºC after removing from PCR machine		*Immediately place on ice or in 4 ºC after removing from PCR machine
		+
		+	*PCR reactions should be set up on ice.
		+
		+	*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

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Revision as of 20:54, 17 October 2007

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

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

• PCR reactions should be set up on ice.

• 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