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Cutting DNA

Cutting DNA. Restriction endonucleases (restriction enzymes) sticky ends blunt ends Nomenclature Eco RI E = genus ( Escherichia ) co = species ( coli ) R = strain I = # of enzyme. Blunt & Sticky ends. Pasting DNA. Complementary ends (sticky ends) H-bond

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Cutting DNA

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  1. Cutting DNA • Restriction endonucleases (restriction enzymes) • sticky ends • blunt ends • Nomenclature • EcoRI • E = genus (Escherichia) • co = species (coli) • R = strain • I = # of enzyme

  2. Blunt & Sticky ends

  3. Pasting DNA • Complementary ends (sticky ends) H-bond • Ligase forms phosphodiester bond to seal strands together.

  4. A plasmid vector for cloning • Contains an origin of replication, allowing for replication independent of host’s genome. • Contains Selective markers: Selection of cells containing a plasmid • twin antibiotic resistance • blue-white screening • Contains a multiple cloning site (MCS) • Easy to be isolated from the host cell.

  5. Plasmid vectors

  6. Expression vector

  7. Isolation of cloning vector (bacterial plasmid) & gene-source 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 How to clone DNA

  8. 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 Screening of the clone

  9. lacZ insert functional enzyme nonfunctional enzyme X-gal product X-gal product Blue/White Color Screening lacZ

  10. Selecting Colonies with Recombinant Plasmids

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