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amp" gene -lacZ gene -Gene of interest Plasmid DNA - Unique restriction - Chromosomal DNA site Origin of- replication from human cells Cut the DNAS with the same restriction enzyme. Mix the DNAS together. Allow time for sticky ends to base-pair. Add DNA ligase to covalently link the DNA backbones. Gene of interest Another fragment of chromosomal DNA Recircularized Recombinant Recombinant or or vector vector vector Mix DNA with many E.coli cells that don't already have a plasmid. Treat the cells with agents that make them permeable to DNA. Note: This shows a bacterial cell that has taken up a plasmid carrying the gene of interest. Other bacterial cells would have other recombinant Recircularized vector without an insert; B-galactoside is made and converts X-Gal to a blue color. vectors or a recircularized vector. Plate cells on media containing X-Gal, IPTG, and ampicillin. Incubate overnight. Recombinant vector with an insert; Blue colony White colony B-galactosidase is inactive because of the inserted DNA. Each bacterial colony is derived from a single cell that has taken up a single plasmid. Therefore, all the cells in a colony are genetically identical. Cells that do not take up a plasmid cannot grow and form colonies because they are killed by ampicillin. FIGURE 21.2 The steps in gene cloning. Note: X-Gal refers to the colorless compound 5-bromo-4-chloro-3-indolyl- B-D-galactoside. It is converted by B-galactosidase into a blue dye. IPTG is an abbreviation for isopropyl-ß-D-thiogalactopyranoside, which is a nonmetabolizable lactose analog that induces the lac promoter. ONLUNE

amp" gene -lacZ gene -Gene of interest Plasmid DNA - Unique restriction - Chromosomal DNA site Origin of- replication from human cells Cut the DNAS with the same restriction enzyme. Mix the DNAS together. Allow time for sticky ends to base-pair. Add DNA ligase to covalently link the DNA backbones. Gene of interest Another fragment of chromosomal DNA Recircularized Recombinant Recombinant or or vector vector vector Mix DNA with many E.coli cells that don't already have a plasmid. Treat the cells with agents that make them permeable to DNA. Note: This shows a bacterial cell that has taken up a plasmid carrying the gene of interest. Other bacterial cells would have other recombinant Recircularized vector without an insert; B-galactoside is made and converts X-Gal to a blue color. vectors or a recircularized vector. Plate cells on media containing X-Gal, IPTG, and ampicillin. Incubate overnight. Recombinant vector with an insert; Blue colony White colony B-galactosidase is inactive because of the inserted DNA. Each bacterial colony is derived from a single cell that has taken up a single plasmid. Therefore, all the cells in a colony are genetically identical. Cells that do not take up a plasmid cannot grow and form colonies because they are killed by ampicillin. FIGURE 21.2 The steps in gene cloning. Note: X-Gal refers to the colorless compound 5-bromo-4-chloro-3-indolyl- B-D-galactoside. It is converted by B-galactosidase into a blue dye. IPTG is an abbreviation for isopropyl-ß-D-thiogalactopyranoside, which is a nonmetabolizable lactose analog that induces the lac promoter. ONLUNE

Biochemistry
Biochemistry
6th Edition
ISBN:9781305577206
Author:Reginald H. Garrett, Charles M. Grisham
Publisher:Reginald H. Garrett, Charles M. Grisham
Chapter28: Dna Metabolism: Replication, Recombination, And Repair
Section: Chapter Questions
Problem 17P
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Explain the role of the gene that is the selectable marker gene in this experiment.

amp" gene -lacZ gene -Gene of interest Plasmid DNA - Unique restriction - Chromosomal DNA site Origin of- replication from human cells Cut the DNAS with the same restriction enzyme. Mix the DNAS together. Allow time for sticky ends to base-pair. Add DNA ligase to covalently link the DNA backbones. Gene of interest Another fragment of chromosomal DNA Recircularized Recombinant Recombinant or or vector vector vector Mix DNA with many E.coli cells that don't already have a plasmid. Treat the cells with agents that make them permeable to DNA.
Transcribed Image Text:amp" gene -lacZ gene -Gene of interest Plasmid DNA - Unique restriction - Chromosomal DNA site Origin of- replication from human cells Cut the DNAS with the same restriction enzyme. Mix the DNAS together. Allow time for sticky ends to base-pair. Add DNA ligase to covalently link the DNA backbones. Gene of interest Another fragment of chromosomal DNA Recircularized Recombinant Recombinant or or vector vector vector Mix DNA with many E.coli cells that don't already have a plasmid. Treat the cells with agents that make them permeable to DNA.
Note: This shows a bacterial cell that has taken up a plasmid carrying the gene of interest. Other bacterial cells would have other recombinant Recircularized vector without an insert; B-galactoside is made and converts X-Gal to a blue color. vectors or a recircularized vector. Plate cells on media containing X-Gal, IPTG, and ampicillin. Incubate overnight. Recombinant vector with an insert; Blue colony White colony B-galactosidase is inactive because of the inserted DNA. Each bacterial colony is derived from a single cell that has taken up a single plasmid. Therefore, all the cells in a colony are genetically identical. Cells that do not take up a plasmid cannot grow and form colonies because they are killed by ampicillin. FIGURE 21.2 The steps in gene cloning. Note: X-Gal refers to the colorless compound 5-bromo-4-chloro-3-indolyl- B-D-galactoside. It is converted by B-galactosidase into a blue dye. IPTG is an abbreviation for isopropyl-ß-D-thiogalactopyranoside, which is a nonmetabolizable lactose analog that induces the lac promoter. ONLUNE
Transcribed Image Text:Note: This shows a bacterial cell that has taken up a plasmid carrying the gene of interest. Other bacterial cells would have other recombinant Recircularized vector without an insert; B-galactoside is made and converts X-Gal to a blue color. vectors or a recircularized vector. Plate cells on media containing X-Gal, IPTG, and ampicillin. Incubate overnight. Recombinant vector with an insert; Blue colony White colony B-galactosidase is inactive because of the inserted DNA. Each bacterial colony is derived from a single cell that has taken up a single plasmid. Therefore, all the cells in a colony are genetically identical. Cells that do not take up a plasmid cannot grow and form colonies because they are killed by ampicillin. FIGURE 21.2 The steps in gene cloning. Note: X-Gal refers to the colorless compound 5-bromo-4-chloro-3-indolyl- B-D-galactoside. It is converted by B-galactosidase into a blue dye. IPTG is an abbreviation for isopropyl-ß-D-thiogalactopyranoside, which is a nonmetabolizable lactose analog that induces the lac promoter. ONLUNE
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