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Cutting DNA Restriction endonucleases (restriction enzymes) • sticky ends • blunt ends Nomenclature • • • • • EcoRI E = genus (Escherichia) co = species (coli) R = strain I = # of enzyme Blunt & Sticky ends Pasting DNA Complementary ends (sticky ends) H-bond Ligase forms phosphodiester bond to seal strands together. A plasmid vector for cloning 1. Contains an origin of replication, allowing for replication independent of host’s genome. 2. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening 3. Contains a multiple cloning site (MCS) 4. Easy to be isolated from the host cell. Plasmid vectors Expression vector How to clone DNA Isolation of cloning vector (bacterial plasmid) & genesource DNA (gene of interest) Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase Introduction of cloning vector into cells (transformation by bacterial cells) Cloning of cells (and foreign genes) Identification of cell clones carrying the gene of interest Screening of the clone The medium in this petri dish contains the antibiotic Kanamycin The bacteria on the right contain Kanr, a plasmid that is resistant to Kanamycin, while the one on the left has no resistance Note the difference in growth Blue/White Color Screening lacZ lacZ functional enzyme X-gal product insert nonfunctional enzyme X-gal product Selecting Colonies with Recombinant Plasmids