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

iGEM Project name 1

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