Team:Brown-Stanford/Lab/Protocols/PhusionPCR

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

1. 98˚, 30 seconds - initial denaturation
2. 98˚, 10 seconds - denaturation
3. 72˚, 4 minutes - annealing and extension
4. Repeat from Step 2 32x
5. 72˚, 10 minutes - final extrension/cleanup
6. 4˚, hold indefinitely