IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/08/13

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assembled assemblies 2, 11, 12, 13, 27, 28, 22, 23, 24, 25

1. Digestion using standard UBC iGEM digestion protocol

  • Prefix samples (cut with EcoRI and SpeI)
  • BBa_J23100
  • Lock 1 (Amp)
  • Key 1 (Amp)
  • ASEM 21
  • Suffix samples (cut with XbaI and PstI)
  • ASEM 17
  • ASEM 18
  • ASEM 19
  • ASEM 20
  • BBa_B0014
  • BBa_E0022
  • BBa_E0034
  • BBa_J31001
  • BBa_J61046


  • Backbone cut (cut with PstI and EcoRI)

pSB1C3 pSB1A3


2. Ligation using standard UBC iGEM digestion protocol

  • ligation into pSB1C3':
  • i) inserts
  • ASEM 02 (1.00μL)
  • ASEM 11 (7.09μL)
  • ASEM 12 (9.32μL)
  • ASEM 13 (1.10μL)
  • ASEM 27 (7.89μL)
  • ASEM 28 (8.83μL)
  • ii) reaction mixture
  • vector: 2μL
  • insert: please see volume in parathesis above
  • water: 14μL - insert volume
  • 10x buffer: 2μL
  • ligase: 1μL

--

  • ligation into pSB1A3':
  • i) inserts
  • ASEM 22 (10.42μL)
  • ASEM 23 (9.51μL)
  • ASEM 24 (9.94μL)
  • ASEM 25 (10.41μL)
  • ii) reaction mixture
  • vector: 2μL
  • insert: please see volume in parathesis above
  • water: 14μL - insert volume
  • 10x buffer: 2μL
  • ligase: 1μL

Overnight ligation in cold (ice) water bath

transformed 10μL of each ligation mix





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