Plasmid DNA Isolation and Characterization by Electrophoresis

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. 

Select and isolate a natural DNA to serve as the carrier (sometimes called vehicle or vector) for the foreign DNA. 

Cleave both strands of the DNA carrier with a restriction endonuclease. 

Prepare and insert the foreign DNA fragment into the vehicle. This produces a recombinant or hybrid DNA molecule. 

Introduce the hybrid DNA into a host organism (usually a bacterial cell), where it can be replicated. This process is called transformation. 

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