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

iGEM Project name 1

Main project page Previous entry      Next entry

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