From 2007.igem.org

---

Revision as of 20:57, 17 October 2007

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

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

• PCR reactions should be set up on ice.

• Add the polymerase last and carefully.

• DMSO is used only for high GC content