Berkeley LBL/PCRextaq
From 2007.igem.org
(Difference between revisions)
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