iGEM 101: Session 5 3/26/15Jarrod Shilts 3/29/15Ophir Ospovat 5a. DNA Ligation Seal fragments of DNA together Formation of phosphoester bonds 5 PO4 of one strand and 3 OH of other strand Complementary sticky ends hold strands in position Vector and insert DNA Ligase T4 DNA Ligase High efficiency ATP dependent Works on blunt ends (but less efficient than sticky end ligation) Temperature dependent Higher temperature breaks H-bonds 37 o C highest activity Room temperature good for sticky ends Cool temperature (~16 o C) good for blunt ends Ligation Reaction Buffer ATP (degraded by freeze-thaw!) Mg2+ salt Tris DTT (keeps enzyme in reduced form) DNA One part vector Three parts insert Ligase Water 5b. Transformation Uptake of foreign DNA Natural transformation (Bacillus) and chemical transformation (Escherichia) Competent cells: M 2+ treated Steps Treat cells for competence Induce DNA entry into cells (heat shock) Outgrowth Selection on plates Liquid culture Cell competence Natural receptors on membrane Uptake induced by stress Restricted to certain species Chemical treatment Offset negative charges Create holes in membrane ? Competence lost if not kept cold Variable degree of competence Strain used Preparation protocol Handling of cells DNA Uptake Heat shock Incubate on ice Exactly 30 seconds at 42 o C Briefly on ice again Electroporation Briefly expose to electric pulse Disrupts membrane and introduces electrical gradient Outgrowth Grow cells in non-selective media Recovery from shock Time to synthesize antibiotic resistance genes Mixed importance Can improve efficiency several-fold Less needed with ampicillin Plating Appropriate antibiotic resistance in plate media Check how old plate is Pre-warm plates while doing outgrowth Plate culture Swirl Beads Spreader Absorption time Incubate 37 o C Make liquid cultures of single colonies