BIOL 2131 Study Guide - Final Guide: Multiple Cloning Site, Lac Operon, Selectable Marker
16 May 2013
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Competency- ability to take up dna: addition of chemicals followed by heat and cold treatments or electric pulses. Luria broth (lb) or lb agar: coli normally can"t grow in ampicillin and can"t break down x-gal. Plasmid pbluescript+ (pbs +) contain 2 genes: ampr (ampicillin resistance- selectable marker , transformed bacteria grow in ampicillin, dark blue colonies (b-galactosidase produces lacz (n terminal region of b- galactosidase 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 cacl2 method- 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.
