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