IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/06/16

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pBAD Strong Mutations As only one colony was produced when using the standard conditions, will try using a lower annealing temperature to produce more colonies. 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 66.5°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