The LacZ Protocol
LacZ is a bacterial gene that produces the enzyme beta-galactosidase, which breaks down lactose into glucose and galactose. The enzyme also cleaves the compound X-gal, which produces a blue color that molecular biologists use as a marker or reporter to track the presence of the LacZ gene in cells.
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Transfection
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Scientists link the LacZ gene with a gene of interest, such as insulin, with a fragment of DNA called a plasmid. They use chemical or electrical transfection methods to get the plasmid into cells. The cells are assayed for LacZ expression of beta-galactosidase, which will also mark the presence of the insulin gene.
fixation
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After transfection, the cells are grown on a plastic petri dish. For the assay, the growth medium is removed and cells are washed with phosphate buffered saline (PBS). A fresh fixation solution of formaldehyde, methanol and glutaraldehyde is used to fix the cells for two minutes. The fixative is removed and the cells are washed again with PBS.
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X-gal
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A fresh staining solution that contains X-gal, potassium ferri-cyanide and potassium ferro-cyanide is added to the cells overnight. The cells are washed again with PBS, and if the LacZ gene is present, the beta-galactosidase will have cleaved the X-gal and the cells will be blue.
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