385-386) Technique Overview: Cloning a Human Gene in a Bacterial Plasmid Most protein-coding genes exist in only one copy per genome-something on the order of one part per million of DNA-so the ability to clone such rare DNA fragments is extremely valuable.” (Campbell & Reece, AP Edition Biology Seventh Edition, pp. Alternatively, a protein with medical uses, such as human growth hormone, can be harvested in large quantities from bacterial cultures carrying the cloned gene for the protein. For example, a resistance gene present in one crop species might be cloned and transferred into plants of another species. Researchers can isolate copies of a cloned gene from bacteria for use in basic research or to endow an organism with a new metabolic capability, such as pest resistance. Cloned genes are useful for two basic purposes: to make many copies of a particular gene and to produce a protein product. Therefore the clone of cells contains multiple copies of the gene. Because the dividing bacteria replicate the recombinant plasmid and pass it on to their descendants, the foreign gene is “cloned” at the same time. The plasmid is returned to a bacterial cell, producing a recombinant bacterium, which reproduces to form a clone of identical cells. The resulting plasmid is now a recombinant DNA molecule, combining DNA from two sources. For cloning genes or other pieces of DNA in the laboratory, a plasmid is first isolated from a bacterial cell, and then the foreign DNA is inserted into it. Bacterial plasmids are relatively small, circular DNA molecules that replicate separate from a bacterial chromosome. One common approach uses bacteria (most often, Escherichia coli) and their plasmids. Selection from the Campbell and Reece, AP Edition Biology, Seventh Edition, 2005 “Most methods for cloning pieces of DNA in the laboratory share certain general features. This topic part has two sections: Content Tutorial & Animations. This is Part A, Constructing Recombinant DNA, under the module topic, Molecular Cloning. be able to make sterilized selective liquid media for growing up one of the bacterial colonies containing the gene of interest to produce large quantities of the desired plasmid DNA.be able to isolate bacterial colonies carrying the plasmid by analyzing the agarose gel and determine samples containing the gene of interest.be able to make an agarose gel, load the gel with your PCR reaction samples, and conduct agarose gel electrophoresis of samples.be able to screen resistant colonies for the gene of interest by amplifying individual bacterial colony DNA through Restriction Enzyme Digestion.be able to plate transformed bacterial cells on selective plates and identifyresistant colonies.be able to make selective media plates for plating and growing transformed bacterial cells (antibiotic selective or X-gal).be able to perform a bacterial transformation reaction to transform competent bacterial cells with recombinant plasmid DNA.be able to recombine a digested DNA fragment and plasmid vector in a ligation reaction to make recombinant plasmid DNA carrying a gene of interest.be able to conduct a restriction digestion to prepare DNA fragments and vectors for the construction of recombinant DNA.be able to identify compatible restriction sites for cloning.Upon completion of this module topic, you will: