Molecular cloning experiments

Experiment 14 introduced students to some principles and techniques involved in recombinant DNA research. Specifically, the experiment outlined the replication, isolation, and analysis of bacterial plasmid vehicles for molecular cloning experiments. This experiment describes another tool that is essential in hybrid plasmid construction and analysis-restriction enzyme action. The procedures introduced here have also found widespread use in the analysis and characterization of all DNA molecules. 

Phosphoinositase C (i.e. phosphoinositide-specific phospholipase C [PLC]) enzymes are found in the vast majority of mammalian cells. Molecular cloning of these enzymes, analysis of their predicted amino acid sequences and immunological cross-reactivity indicate that at least three major forms of the enzyme exist PLC-/I, -8 and -y. Each of these enzyme types is encoded by a distinct gene. More recent experiments using the polymerase chain reaction and molecular cloning have revealed even greater enzyme di-... 

Extrachromosomal DNA molecules called plasmids are harbored in some strains of E. coli. The normal copy number of the plasmids is small, between 2 and 10 however, if these strains of E. coli are grown in the presence of chloramphenicol, up to 3000 copies may be replicated per cell. Plasmid DNA has been demonstrated to be a useful vehicle in molecular cloning. This experiment describes a method for the growth of E. coli and amplification of the ColEl plasmids. The plasmids will be isolated from E. coli cells by one of two methods, a large-scale boiling method or a microscale alkaline lysis method. The DNA plasmids will be measured for molecular size by agarose electrophoresis. 

L. Perez-Pons and E. Querol, Biochem. Educ. 24, 54-56 (1996). A laboratory experiment illustrating basic principles of DNA cloning and molecular biology techniques. J. Sambrook, E. Fritsch, and T. Maniatis, Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory (Cold Spring Harbor, NY), Vols. I, II, and III, pp. 1.21-1.52. Techniques in recombinant DNA. 

The hypothesis that there are subtypes of a2 receptors emerged from pharmacologic experiments and molecular cloning. It is now known that there are three subtypes of 2 receptors, termed < 2a, 2B, and i2c, that are products of distinct genes. 

This article presents a brief review of the propenies of enzymes used in preparing DNA and RNA probes for hybridization. Included are reaction conditions typically employed and examples of types of experiments generally performed with different probes. More comprehensive protocols may be found in Molecular Cloning A Laboratory Manual (1) [Pg.116]

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