We used [http://www.neb.com/nebecomm/products/productM0531.asp Phusion High Fidelity PCR Master Mix] from New England Biolabs.

Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (98 degrees C). All components should be mixed and centrifuged prior to use. It is important to add Phusion Master Mix last in order to prevent any primer degredation.

For 50 uL reaction:

10 uM forward primer	2.5 uL
10 uM reverse primer	2.5 μL
Template DNA	1 uL
Nuclease-free water	to final volume (including mix) of 50 μL
2X Phusion Master Mix	25 μL

Transfer PCR tubes from ice to a PCR machine with the block preheated to 98 degrees C and begin thermocycling.

Thermocycing conditions for a routine PCR:

Initial denaturation	98 degrees C	30 seconds
25-35 cycles	98 degrees C	5-10 seconds
	45-72 degrees C	10-30 seconds
	72 degrees C	15-30 seconds
Final extension	72 degrees C	5-10 minutes
Hold	3 degrees C