IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/06/18

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pBAD Strong Mutations

  • To try and get more PCR product, will put in more DNA template and perform a step down PCR

PCR Mixture

  • 10ul 5X Phusion HF Buffer
  • 1uL 10mM dNTP mix
  • 27.4uL diH2O
  • 2.5uL of 10uM Positive Forward Primer
  • 2.5uL of 10uM Reverse Primer
  • 6.1uL of 1/2000 dilution pBAD DNA Template
  • Add 0.5uL DNA Polymerase just before starting the PCR

PCR Conditions - Lower Annealing Temperature

  • 98°C - 30sec
  • 98°C - 10sec
  • 60°C (-0.5°C every cycle) - 30 sec
  • 72°C - 60sec
  • Repeat steps 2-4 for 25 cycles
  • 72°C -10 min
  • 4°C - hold

Gel PCR Confirmation

  • Run a gel of 10uL of PCR product
  • Used standard gel conditions of 1% agarose, 0.5X TBE buffer, 1uL CYBR Safe, 100V, 1hour
  • A very faint band was seen on the gel
  • No PCR Product was seen on the gel