IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/06/17

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Standardizing plasmid Concentration to 100ng/μL

  • Created a 100ng/μL concentration sample for each of the following plasmids:
Name Part Number
RFP E1010
Assembly 1
pBAD BBa_I13453
pBAD Positive
pBAD Negative 1
pBAD Negative 2
Chlor construction BBa_P1010
J23100 + rbs BBa_J61100
RBS B0034
YFP E0430
Terminator BBa_B0014
J23100 J23100
  • The reminding volume of the plasmids are stored back into the fridge
  • The new 100ng/μL concentration samples are kept separately inside the same box

pBAD Strong Mutations

  • Site-Directed mutagenesis was performed with a lower annealing temperature to try and get more PCR product
  • An annealing temperature of 53°C was chosen as it is 5°C lower then the Tm given by IDT, the company that made the primers. Previously the Tm was calculated using the Finnzyme website suggested by the Mutagenesis Kit

PCR Mixture

  • 10ul 5X Phusion HF Buffer
  • 1uL 10mM dNTP mix
  • 27.9uL diH2O
  • 5uL of 5uM/uL Positive Forward Primer
  • 5uL of 5uM/uL Reverse Primer
  • 0.6uL of 1/10000 pBAD DNA Template
  • Add 0.5uL DNA Polymerase just before starting the PCR

PCR Conditions - Lower Annealing Temperature

  • 98°C - 30sec
  • 98°C - 10sec
  • 53°C - 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