IGEM:MIT/2005/Thursday, June 9th

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MIT iGEM 2005 Laboratory "Bootcamp" Day 4 (run by Natalie Kuldell and Kate Bacon Schneider)

I. Ligations

  • Background Information:
    • Ligase is the enzyme that can catalyze the formation of the phosphodiester bond between two nucleotides
    • ATP dependent reaction
    • Molar ratio of insert: backbone should be 2:1 or 3:1-->want to favor insert ends finding vector ends
    • Want to do control with vector DNA alone-->growth after transformation tells you if the vector religated (or was uncut in the first place)
    • Typical reaction conditions (20 µL total volume):
      • water
      • 1 µL vector (backbone)
      • µL insert (part)
      • 10X ligation buffer-->working concentration of 1X
      • 0.5 µL ligase
    • Run reaction for 10-15 minutes at 15˚C
      • low temperature favors hydrogen bonding of sticky ends to each other, and reduces molecular motion so that ends can "find" each other in solution)
  • Exercise:
    • Students ligate their purified vector and insert along with appropriate controls

II. Transformations

  • Background Information:
    • transformation is a method for introducing plasmid DNA into (bacterial) cells
    • E. coli is not naturally able to take up DNA from its environment, but other bacterial species are
    • many ways to make E. coli "competent"—or able to take up DNA from the cellular environment—including electroporation and CaCl2 treatment
    • Making cells "competent" involves a weakening of the cell membrane-->therefore cells are fragile (so keep on ice)
    • Basic protocol involves:
      • incubating DNA and cells together (allows DNA to interact with cell surface
      • heat shock cells (usually 37-42˚C)-->cells take up DNA
      • ice (close up pores of membrane)
      • outgrowth in rich media (allows cells to "recover" from treatment and to express the gene that confers resistance to antibiotic on backbone
      • plating on selective media (chosen such that only cells that have taken up ligated plasmid will grow)
  • Exercise:
    • Students set up three transformations of 7.02 cloning strain (AG1111, CaCl2 competent): vector + insert ligation, vector alone ligation, no DNA ligation.
    • Cells were plated on LB Amp and grown O/N @ 37˚C