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Team:Brown-Stanford/Lab/Protocols/PhusionPCR
From 2011.igem.org
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=='''Phusion® PCR'''== | =='''Phusion® PCR'''== | ||
| - | (Taken from the [http://www.finnzymes.com/pdf/f530sl_phusion_datasheet_2_2_low.pdf Phusion® High-Fidelity DNA Polymerase Protocol] | + | (Taken from the [http://www.finnzymes.com/pdf/f530sl_phusion_datasheet_2_2_low.pdf Phusion® High-Fidelity DNA Polymerase Protocol]; this protocol was edited for amplification of [http://partsregistry.org/Part:BBa_K656013 Part BBa_K656013]) |
'''Reagents for 20 µL reactions''' | '''Reagents for 20 µL reactions''' | ||
| + | *11.8µL Nuclease-Free Water | ||
* 4µL 5x Phusion® HF Buffer | * 4µL 5x Phusion® HF Buffer | ||
* .4 µL 10 mM dNTP | * .4 µL 10 mM dNTP | ||
| - | * | + | * 1 µL primer A (.5µM concentration recommended) |
| - | * | + | * 1 µL primer B (.5µM concentration recommended) |
| - | * | + | * 1 µL template DNA |
* .6µL DMSO (optional; recommended for GC-rich amplicons) | * .6µL DMSO (optional; recommended for GC-rich amplicons) | ||
* .2µL Phusion® DNA Polymerase | * .2µL Phusion® DNA Polymerase | ||
| + | |||
| + | |||
| + | '''Thermocycle''' | ||
| + | #98˚, 30 seconds - initial denaturization | ||
| + | # 98˚, 10 seconds - denaturation | ||
| + | # 72˚, 4 minutes - annealing and extension | ||
| + | # Repeat from Step 2 32x | ||
| + | # 72˚, 10 minutes - final extrension/cleanup | ||
| + | # 4˚, hold indefinitely | ||
Revision as of 07:36, 28 September 2011
Phusion® PCR
(Taken from the [http://www.finnzymes.com/pdf/f530sl_phusion_datasheet_2_2_low.pdf Phusion® High-Fidelity DNA Polymerase Protocol]; this protocol was edited for amplification of [http://partsregistry.org/Part:BBa_K656013 Part BBa_K656013])
Reagents for 20 µL reactions
- 11.8µL Nuclease-Free Water
- 4µL 5x Phusion® HF Buffer
- .4 µL 10 mM dNTP
- 1 µL primer A (.5µM concentration recommended)
- 1 µL primer B (.5µM concentration recommended)
- 1 µL template DNA
- .6µL DMSO (optional; recommended for GC-rich amplicons)
- .2µL Phusion® DNA Polymerase
Thermocycle
- 98˚, 30 seconds - initial denaturization
- 98˚, 10 seconds - denaturation
- 72˚, 4 minutes - annealing and extension
- Repeat from Step 2 32x
- 72˚, 10 minutes - final extrension/cleanup
- 4˚, hold indefinitely
