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number3 - 2 lacZ encodes for beta galactosidase-hydrolyzes...

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3) 20.1 (386-388) @ Read "Transformation of E. Coli …" pgs 34-37, Lab Guide Do Lab 6.I, LabBench tutorial, on campbellbiology.com print quiz, bring for stamp A. Describe the uses of engineered plasmids as cloning vectors . B. Summarize the steps of gene cloning described on page 368. amp R lacZ b-galactosidase X-gal hybridization probe denaturation (Fig 20.4) A) -Cloning prepares many copies of a gene of interest for use in sequencing a gene to produce its protein. Cloning Vector: A DNA molecule that carries foreign DNA into a cell to replicate there. -Bacterial plasmids are frequently used as cloning vectors because - easily isolated from bacteria, manipulated to form recombinant plasmids by insertion of foreign DNA in vitro, and then reintroduced into bacterial cells. -DNA reproduces rapidly. B) 1) Isolate bacterial plasmid from E.coli cells. -Isolate the gene of interest form the human cells in the culture. -The bacterial plasmid has 2 genes: 1) ampR = ampicillin resistance
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Unformatted text preview: 2) lacZ- encodes for beta galactosidase-hydrolyzes sugar lactose and synthetic molecular mimic called X-gal . [blue] 2) Plasmid and human DNA are restricted.-Plasmid is cut in the lacZ gene.-Human DNA is cut at many sites.-Each has sticky ends. 3) Mix human DNA fragments with cut plasmids-They base pair [complementary]-DNA ligase seals all fragments together.-Recombinants are created.-Not all DNA is recombinant. 4) The recombinant DNA above is mixed with bacteraia that has a mutation in own lacZ gene making them unable to hydrolize lactose as well.-Cells take up foreign DNA by transformation-Cells are various types of recombinants. 5) Bacteria is placed in solid agar that has ampicillin and X gal so that those with ampR gene will grow. Those would be plasmids. If cells are blue, then those are eliminated, for the blue cells have the lacZ gene and were not cut properly [also plasmids]....
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