BCMB20005 Lecture Notes - Lecture 3: Bunsen Burner, Molecular Cloning, Phosphodiester Bond

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All figures from lecture 3 slides, a willems-jones, march 2017, melbourne university (unless stated) Dna ligase: mechanism of action: formation of phosphodiester bond (3 step process) Transfection/transformation of recombinant dna into a host cell: heat shock method, electroporation method. Selection: restriction mapping logic, blue/white selection (lacz inactivation) Dna and plasmid both been digested with 2 enzymes: ecori (sticky end) and pvuii (blunt end) Genes can be inserted into the plasmid in a specific direction directional cloning. cut with the same restriction enzyme(s) so that the 5" a(cid:374)d the 3" e(cid:374)ds match up. In dna cloning strategy both gene of interest and plasmid should be. Dna ligase reforms phosphodiester (cid:271)o(cid:374)ds (cid:271)etwee(cid:374) 3"- h(cid:455)dro(cid:454)(cid:455)l a(cid:374)d 5"phoosphate whi(cid:272)h needs: atp-dependent, 3 main steps. Transfection (transformation) (cid:862)the pro(cid:272)ess of i(cid:374)trodu(cid:272)i(cid:374)g dna i(cid:374)to (cid:272)ells(cid:863) Can be used to introduce foreign dna into eukaryotic cells. Two common procedures: heat shock method (cacl2 method)

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