User:Matt Hartings/Notebook/Photosynthesis/2012/07/11

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Objective

Start cloning sperm whale myoglobin gene into pQE-80 vector

PCR Cloning

  1. Prep primers
    1. Forward Primer (5'-AACAGGATCCGTTCTGTCTGAAGGTGAATGGCAG-3') 0.3mg MW=10546.9g/mol
      1. Add 284uL of autoclaved water to forward primer to make 100uM solution
    2. Reverse Primer (5'-AAGCTTACCCTGGTAACCCAGTTCTTTGTA-3') 0.32mg MW=9132g/mol
      1. Add 350uL of autoclaved water to reverse primer to make 100uM solution
  2. Prep PCR tube for reaction
    1. 37uL of autoclaved water
    2. 10uL of 10X Pfusion HF buffer
    3. 1uL of dNTPs
    4. 1uL forward primer (meant to use 0.5uL)
    5. 1uL reverse primer (meant to use 0.5uL)
    6. 1uL of WT swMb DNA
    7. 0.5uL of Pfusion HF enzyme
    8. 50uL of wax
  3. Place in PCR machine
  4. Run DEL program. (Tamra had edited an older program)



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