IGEM:Indian Institue of Technology Madras/2009/Notebook/PLASMID - Plasmid Locking Assembly for Sustaining Multiple Insert DNA/2009/09/28

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  • OD v/s Conc expt: (DH5aplha; K12Z1; DH5aplha(P2 Plasmid); lacI- cells(P2 plasmid))
  • 3ml inoculum prepared.
  • 1.5ml added to Flasks
  • OD and conc were measured every 30 mins.
  • OD was calculated after resspending the cell pellet in Saline.
  • AHL confirmation:
  • AHL experiment: (DH5alpha; P2 in LacI- cells; T9005 cells)
  • Incubate all 3 cell types.
  • 2 mixtures made:
  • 1) 2ml T9005+ 2ml LacI- + 2 ml 2xyt broth -----> with Amp
  • 2) 2ml T9005+ 2ml DH5aplha + 2 ml 2xyt broth --> without Amp
  • GFP expt:
  • inoculate DH5alpha cells in 5ml culture.
  • Add IPTG 3 hrs after incubation
  • Measure CFP after 5 hrs
  • The following was done as controls for restriction; ligation; Alkaline phosphatase treatment; and Transformation.
  • Restricted J23119(1A3) with S/P.
  • Restricted K156011(PCR) with X/P.
  • Alkaline Phosphatase (AP) of half of J23119(S/P) and half of K156011(X/P)
  • Following ligations performed: (AP = alkaline phosphatase treated)
  • C1) J23119(AP) + K156011;
  • C2) J23119(AP) + K156011(AP);
  • C3) J23119 + K156011(AP);
  • C4) J23119 + K156011;
  • C5) J23119(AP)
  • C6) J23119
  • Transformations of:
  • C1 through C6; C7(unligated J23119(S/P)); C8(unligated J23119(AP)(S/P))


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