IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/08/11

iGEM Project name 1

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cPCR cPCRed 1.33ul of each liquid culture (ASEM10, 11, [LOX]R, Cre-LVA) to verify construct note: each assembly were innoculated in triplicate 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: 18.8 liquid culture: 1.33 Taq polymerase: 0.5 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 of cPCR ASEM10, 11, [LOX]R, Cre-LVA Expected bands were obtained for ASEM10, [LOX]R, and Cre-LVA. Miniprep and made glycerol stocks of selected ones. Miniprep of ASEM10, [LOX]R, and Cre-LVA successfully performed using the standard UBC iGEM Raf's Miniprep Protocol diluted ASEM10, [LOX]R, and Cre-LVA to 100μg/μL stocks and sent for sequencing