IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/08/20

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Construct verification via cPCR

 * negative controls from yesterday look good (no growth); thus the antibiotics worked!
 * no growth for loxR-pSB1C3 triplicate number 3; thus used a colony from spot plate instead

cPCR

 * cPCRed ~1ul of each liquid culture via standard UBC iGEM cPCR protocol (using autoclaved wooden sticks)


 * The cultures were: (each in triplicate)
 * pSB1C3 + LoxR
 * pSB1A3 + LoxR
 * pSB1C3 + Cre-LVA
 * pSB1A3 + Cre-LVA

Forward (FW): VF2 Reverse (RE): VR2
 * primers


 * reagents for each reaction (μL)
 * 10x reaction buffer: 2.5
 * 10μM Forward primer: 1.25
 * 10μM Reverse primer: 1.25
 * 10mM dNTP: 0.5
 * sdH20: 18.3
 * liquid culture: ~1 (transferred using autoclaved wooden stick)


 * PCR steps [temperature | time ]
 * Initial denaturation: 94°C | 120s
 * Denaturation: 94°C | 30s
 * Annealing: 56°C | 30s
 * Extension: 72°C | 72s
 * Final extension: 72°C | 216s
 * Step 2-4 repeated 30 cycles


 * Gel electrophoresis

Correct bands were observed, suggesting that the constructs were successful. One correct band from each construct will be miniprepped (Aug 20)


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