Microbiology and Immunology 2500A/B Lecture Notes - Lecture 32: Dna Ligase, Recombinant Dna, Sticky And Blunt Ends
10 Jan 2017
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Dna ligase: dna ligase = the glue for dna, will reseal compatible sticky ends and much less ef ciently, blunt ends. Will do it but less ef ciently: having sticky overhang ends lets an intermediate form with base pairing, requires an energy source (atp), ligase requires 5" phosphates for a ligase to be able to work. Steps in cloning: digest 100ng of yfg and a plasmid vector with the same restriction enzyme or one that gives compatible overhanging sequences. Cut the plasmid and our favourite gene to leave sticky ends: purity your fragments - often by electrophoresis. Incubate vector (plasmid) + insert (yfg) together in the presence of dna ligase and atp: the sticky ends anneal (hybridize). Ligase seals the ends with covalent phosphodiester bonds. The process by which cells take up dna from their environment. Natural property of some bacteria - e. coli must be treated with chemicals to do it. Some of the e. coli will take up the plasmid.
