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

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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

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: 18.8
 * 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