IGEM:MIT/2005/Thursday, June 9th
From OpenWetWare
Jump to navigationJump to search
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