IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/08/05

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Repeat of cPCR of ASEM 1, 2, 19, 11, 12 to verify constructs

  • performed using standard UBC iGEM cPCR protocol (cultures were 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
  • Taq polymerase: 0.5
  • sdH20: 18.8
  • liquid culture: 1.0 (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

  • One correct band was observed for each of ASEM 1, ASEM12, Key1B, and Control Lock constructs. The culture corresponding to the correct band was be miniprepped using the standard UBC iGEM Raf's Alkaline Lysis prorotcol and diluted to 100ng/μL working solutions.
  • No bands at al for ASEM 2 triplicates
  • ASEM 10 bands were not of the correct size


preparation for experiments tomorrow

  • innoculated in triplicate using standard UBC iGEM protocols ASEM16 to ASEM for the cPCR tomorrow





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