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Transformation Lab

Circular sequences of DNA that can be incorporated into a bacterial host genome. Transformation Lab. What makes them so special??. What are plasmids?. They contain: Origin of Replication Selection Marker Gene (Amp) Color Marker for easy selection. pGREEN Plasmid.

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Transformation Lab

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  1. Circular sequences of DNA that can be incorporated into a bacterial host genome. Transformation Lab What makes them so special?? What are plasmids? • They contain: • Origin of Replication • Selection Marker Gene (Amp) • Color Marker for easy selection

  2. pGREEN Plasmid

  3. Beta-galactosidase and X-gal • The beta-galactosidase gene encodes a protein that normally cleaves the disaccharide sugar lactose into galactoseand glucose. However, the enzyme will also cleave many other molecules that contain structures resembling the galactose-glucose link. Researchers have developed several chemicals that, when cleaved by beta-galactosidase, produce colored products. One of these color indicators is the chemical 5-bromo-4-chloro-3-indolyl-β-D- galactoside. Fortunately, the chemical is usually referred to by its common name, X-gal. X-gal is colorless, but when it is cleaved by beta-galactosidase, one of the products is dark blue. Therefore, if you grow bacteria that produce beta-galactosidaseon media containing X-gal, the colonies will be bright blue.

  4. pBLU Plasmid

  5. Cloning • Cloning really means inserting DNA so that it is propagated in that cell and its progeny. • In order to do this in bacteria, the “gene of interest” has to be inserted into the plasmid (which is used as a vector). This is done in several steps.

  6. Restriction Enzymes • Restriction enzymes Cut DNA at specific sequences. • DNA Ligase reattaches that DNA into the plasmid

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