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:


 * 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


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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