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55-213 lab 1 exam review.docx

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University of Windsor
Biological Sciences

Lab 1: Bacterial Transformation - Competency- ability to take up DNA o Addition of chemicals followed by heat and cold treatments or electric pulses - Bacterial transformation- cloning (propagate, express and isolate recombinant DNA molecules) and genetic engineering, Applications: plants and animals resistant to diseases - E. Coli are easy to grow- 37C, no special requirements, normal inhabitant of the human colon and soil, easily grown in suspension in liquid media or semi solid media (contain nutrients provided by Luria broth (LB) or LB agar) - E. Coli normally can’t grow in ampicillin and can’t break down X-gal - Plasmid pBluescript+ (pBS +) contain 2 genes: o AmpR (ampicillin resistance- selectable marker ) o lacZ (N terminal region of B- galactosidase o Transformed bacteria grow in ampicillin, dark blue colonies (B-galactosidase produces blue precipitate on enzymatic hydrolysis) - pBS+ contains multiple cloning site (MCS) within lacZ gene inserts foreign genes- disrupted lacZ white colonies (have foreign DNA) - Sterile technique and plating bacteria ensures no foreign DNA is inserted into the MCS - Competent cells prepared via CaCl meth2d- makes holes in bacterial mb/cell wall to allow intake of DNA followed by hot-cold treatment- cold incubation allows DNA to stick to membrane and heat shock (either makes membrane more fluid or holes bigger) allows take up of DNA - Phenotypic delay- recovery period after the hot-cold treatment, also allows the genes to be expressed in the phenotype Plasmid: - Amp resistance is a selectable marker - LacZ encodes for B-galactosidase breaks down bond between galactose and lactose but also X-gal (if present) this releases a blue colour - LacZ gene is a reporter gene- will report if the gene is intact (because it
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