Berkeley LBL/PCRextaq
From 2007.igem.org
< Berkeley LBL(Difference between revisions)
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Add H2O to total volume 50 uL | Add H2O to total volume 50 uL | ||
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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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2. ''Run PCR machine'' | 2. ''Run PCR machine'' | ||
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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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| - | * | + | '''*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 | ||
Latest revision as of 21:01, 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
- 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
