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Isbn-6514-3 microbiology a clinical approach by tony

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Unformatted text preview: ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science GENERATING A RECOMBINANT MOLECULE: STEPS OF A CLONING EXPERIMENT ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science CLONING VECTORS AND CREATING RECOMBINANT DNA There are four types of cloning vectors Plasmids (most commonly used) Bacteriophage Cosmids Artificial chromosomes ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science A PLASMID VECTOR USED FOR CLONING Each type of cloning vector generally has: Self-replicating, origin of replication, ori A selectable marker, a gene whose product allows cells to grow in conditions that would otherwise be lethal ( amp R) A multiple-cloning site (MCS), has the recognition sequence of several unique restriction enzymes Second genetic marker, lacZ’ ( β ¡ galactosidase ) , spans the MCS, used to distinguish cells that contain recombinant plasmids from those containing intact vector ampR lacZ’ ori MCS ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science THE FUNCTION OF THE lacZ’ GENE IN A VECTOR The product of lacZ’ enables cells to cleave a colorless chemical, x-gal, to form a blue compound. ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science CLONING THE SOMATOSTAIN GENE ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science CLONING CELLULAR DNA FRAGMENTS ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach, by Tony Srelkauskas © Garland Science Microbiology: A Clinical Approach © Garland Science INTRODUCING THE RECOMBINANT DNA INTO NEW HOST Most common hosts E. coli : a prokaryotic host S. cerevisiae : a eukaryotic host DNA introduction into microbes Transformation E. coli cells must be specially treated to induce them to take up DNA Simple chemical treatments can make cells competent, or able to take up external DNA Electroporation Bacteria and DNA are mixed together and the mixture is subjected to an electric current The current makes holes in the plasma membrane through which the DNA enters Microinjection of foreign DNA into a fertilized mouse egg The egg is first immobilized by applying suction to the large, blunt, holding pipette Several hundred copies of the gene of interest are then injected into the nucleus of the cell through the tiny end of the micropipette Gene gun can be used to insert DNA-coated “bullets” into a a cell ISBN: 978-0-8153-6514-3 Microbiology: A Clinical Approach,...
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