User:Matt Hartings/Notebook/Photosynthesis/2012/07/11: Difference between revisions

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#Place in PCR machine
#Place in PCR machine
#Run DEL program. (Tamra had edited an older program)
#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 [http://www.neb.com/nebecomm/ManualFiles/manualE0553.pdf  Phusion® High-Fidelity PCR Kit].




Revision as of 08:30, 11 July 2012

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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)
    1. Start with 30 seconds at 98°C on the thermocycler.
    2. Cycle through the following 40 times:
      • 10 seconds at 98°C
      • 30 seconds at 62°C
      • 30 seconds at 72°C
    3. A final extension step of 5 min was done at 72°C.
    4. The thermocycler was then held at 4°C.

Most components for this PCR came from the Phusion® High-Fidelity PCR Kit.



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