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

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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)


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