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cloning - Molecular Cloning 1 Isolate gene from its content...

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Molecular Cloning 1. Isolate gene from its content – most difficult step a. Why can’t you put gene into another? – degradation because not heritable b. Vectors: Use plasmid Lac Z’ – with multiple cloning site: a dense concentration of restriction sites on a vector – want sticky ends. 2. Restriction Digest with appropriate endonuclease to make matching sticky ends 3. Ligation 4. Transformation with Competent Ampicillin Sensitive Cells a. Bacteria w/o plasmid (untransformed) b. Bact w/ plasmid w/o insert c. Bact w/ plasmid w/ insert 5. Blue-White screening a. Add ampicillin + X-gal + IPTG i. Ampicillin kills (a.) – bacteria w/o the plasmid Beta-galactosidase will split X-gal producing a blue dye – this is the bacteria with the plasmid without the insert in Lac Z’ which is constantly producing B-galactosidase because IPTG (by taking the place of allolactose) is constitutively producing B- galactosidase. IPTG is an inducer and is useful because it doesn’t get used up like glucose, so it always allows production of B-gal.
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