INSERTION AND ISOLATION OF PLASMIDS 

Theory:  Plasmids are the vehicle for transferring recombinant DNA into a host cell.  A plasmid that has been manipulated (cut with restriction endonucleases) to contain ligated pieces of DNA from other sources is referred to as a recombinant plasmid.  Some bacteria can spontaneously take up free DNA in a solution and become transformed.  These bacteria are described as competent.  In order to induce transformation competence, bacterial cells are first treated with an ice-cold solution of calcium chloride.  It is believed that the positive calcium chloride binds to the negatively charged DNA strands.  The solution is then heat shocked at high temperature.  This dual treatment opens and then closes the pores of the bacterial envelope rendering it susceptible to penetration by large DNA fragments.  In order to know that the plasmid has been absorbed into the bacteria host cell, one of the genes carried in the plasmid is for a protein that inactivates the antibiotic ampicillin.  Therefore, bacteria that carry such a plasmid are resistant to the antibiotic and will grow when the antibiotic is added to the culture medium. This is an example of antibiotic selection.  Along with this treatment, cultures can be made to have an environment that affects the activation of a gene.  The resistance to ampicillin and the use of genetic switches can be used as controls.

Purpose   |   Procedures   |   Results