Berkeley LBL/PCRphusion

From 2007.igem.org

(Difference between revisions)
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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
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*PCR reactions should be set up on ice.
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*Add the polymerase last and carefully.
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*DMSO is used only for high GC content
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*5x GC Buffer may be used in replacement of HF Buffer for high GC content for Phusion kit

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