From 2007.igem.org

PCR (TaKaRa Ex Taq Polymerase)

1. Set Up PCR Reaction:

      1 uL template DNA

      5 uL Ex Taq Buffer

      4 uL  dNTP mixture (2.5mM each)

      5 uL Primer Mix

      0.5 uL DMSO (optional)

      0.25 uL TaKaRa Ex Taq Polymerase

      Add H2O to total volume 50 uL

• PCR reactions should be set up on ice.

• Add the polymerase last and carefully.

• DMSO is used only for high GC content

2. Run PCR machine

• Use 1 min/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			1 min/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