Team:Macquarie Australia/WetLab/Protocols/PCR

PCR

PCR Mixture: General recipe for PCR is as follows:

• 4 µL of 5x phusion buffer.
• 0.4 µL dNTPs.
• 0.6 µL of DMSO.
• 0.2 µL of polymerase.
• 11.8 µL of water.

To this mixture, add:

• 1 µL of forward primer.
• 1 µL of reverse primer.
• 1 µL of template.
• Total volume = 20 µL

PCR settings: A general program for PCR is as follows:

• Initial denaturation at 98_oC for 30seconds.

Followed by 30 repeats of:

• Denaturation – 98oC for 10seconds.
• Annealing – 60_oC for 10seconds.
• Extension – 72_oC for 2minutes.
• Final extension – 72_oC for 10minutes.