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
  • 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