IGEM:MIT/2005/Thursday, June 9th

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