Start cloning sperm whale myoglobin gene into pQE-80 vector
- Prep primers
- Forward Primer (5'-AACAGGATCCGTTCTGTCTGAAGGTGAATGGCAG-3') 0.3mg MW=10546.9g/mol
- Add 284uL of autoclaved water to forward primer to make 100uM solution
- Reverse Primer (5'-AAGCTTACCCTGGTAACCCAGTTCTTTGTA-3') 0.32mg MW=9132g/mol
- Add 350uL of autoclaved water to reverse primer to make 100uM solution
- Prep PCR tube for reaction
- 37uL of autoclaved water
- 10uL of 10X Pfusion HF buffer
- 1uL of dNTPs
- 1uL forward primer (meant to use 0.5uL)
- 1uL reverse primer (meant to use 0.5uL)
- 1uL of WT swMb DNA
- 0.5uL of Pfusion HF enzyme
- 50uL of wax
- Place in PCR machine
- Run DEL program. (Tamra had edited an older program)
- Start with 30 seconds at 98°C on the thermocycler.
- Cycle through the following 40 times:
- 10 seconds at 98°C
- 30 seconds at 62°C
- 30 seconds at 72°C
- A final extension step of 5 min was done at 72°C.
- The thermocycler was then held at 4°C.
Most components for this PCR came from the Phusion® High-Fidelity PCR Kit.