Hybrid plasmids

The synthetic and plasmid DNAs are mixed and join their sticky ends spontaneously. They are covalently bound together by DNA ligases, when the resulting hybrid plasmid is inserted into bacterial cells. Dilute calcium chloride solutions render the bacterial membranes permeable and allow the passage of ONA into the cells. 

The yields of DNA intake by the cells are, however, very low. The few bacteria containing the hybrid plasmid need to be sorted out. One technique applies a plasmid which encodes for antibiotic-degrading enzymes, e.g for penicillinase and tetracyclinase, and thus... 

Definition of a plasmid Function in amplification of DNA Prokaryotic organisms normally contain small, circular, extrachromosomal DNA molecules called plasmids that can serve as vectors. They can be readily isolated from the bacterium, annealed with the DNA of interest, and reintroduced into the bacterium which will replicate, thus making multiple copies of the hybrid plasmid. 

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. 

K. Timmis, F. Cabello, and S. N. Cohen, Pwc. Natl. Acad. Scl US, 74 4556-4560 (1974). Utilization of Two Distinct Modes of Replication by a Hybrid Plasmid Constructed in vitro from Separate Replicons. 

Hunig bases, polymeric = animated cbloro-methylated ethenylbenzene homopolymers deprotonation with of ketones, 11 or phosphonium salts, 32 removal of acids with, 32 Hybrid plasmids, 243-245 Hydration. See Alkenes or Alkynes Hydrazine hydrazinolysis with of esters, 239-240,331 of phthaloyl-protected amines, 162,163 reduction of ketones with, 97-98,109 Hydrazines, cyclic oxn., N3 extrusion, 35,331 Hydrazones WolfF-Kishner redn. of, 109 —, aryl- indole synth. with, 151-152, 296, 307 —, dialkyl-, lithiated alkylation, 12, 18, 25-26 enantioselective (SAMP-hydrozones), 25-26 ozonolysis of, 26... 

The mode of entry of recombinant DNA molecules into the host cell is dependent upon the nature of the vector. The introduction of a DNA molecule into a bacterium or any other living cell is termed transformation. Certain species of bacteria, e.g. Bacillus, can easily acquire DNA molecules from a surrounding medium since they have developed pertinent mechanisms. However, most species, e.g. E. coll, can take up only small quantities of DNA. With plasmid vectors, a population of E. coli is bathed in cold calcium chloride (50 mmol dm" ) solution containing the hybrid plasmid. This treatment enhances the DNA binding to the bacterial surface. Raising the temperature to 42°C expedites the transport of the DNA into the cell. Although this technique allows the incorporation of plasmids into the bacterium, the uptake is poor (maximum achievable uptake is approximately 1 %). 

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