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Minipreparation of plasmid DNA is a rapid, small-scale isolation of plasmid DNA from bacteria. [20] [21] Commonly used miniprep methods include alkaline lysis and spin-column based kits. [3] [22] It is based on the alkaline lysis method. The extracted plasmid DNA resulting from performing a miniprep is itself often called a "miniprep".
Alkaline lysis is often an initial step in molecular processes. A proper completion of alkaline lysis yields a pure bacterial plasmid. A plasmid is a circular DNA molecule found naturally in bacteria that replicates independently from chromosomal DNA. Plasmids can also less commonly be found in the other two domains: Archaea and Eukarya.
The term plasmid was coined in 1952 by the American molecular biologist Joshua Lederberg to refer to "any extrachromosomal hereditary determinant." [14] [15] The term's early usage included any bacterial genetic material that exists extrachromosomally for at least part of its replication cycle, but because that description includes bacterial viruses, the notion of plasmid was refined over time ...
Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them. The idea arose that different DNA sequences could be inserted into a plasmid and that these foreign sequences would be carried into bacteria and digested as part of the plasmid. That is, these plasmids ...
conjugative - mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population; e.g., F plasmid, many R and some col plasmids. nonconjugative - do not mediate DNA through conjugation, e.g., many R and col plasmids. The pBR322 plasmid is one of the first plasmids widely used as a cloning vector.
Since DNA is a very hydrophilic molecule, it often cannot penetrate through the bacterial cell membrane. Therefore, it is necessary to make bacteria competent in order to internalize DNA. This may be accomplished by suspending bacteria in a solution with a high calcium concentration, which creates tiny holes in the bacterium's cells [citation ...
The other strand of the plasmid, the strand that was not nicked by the relaxase, is a template for further synthesis by DNA polymerase. [ 17 ] Once the relaxase reaches the upstream section of the oriT again where there is an inverted repeat , the process is terminated by reuniting the ends of the plasmid and releasing a single-stranded plasmid ...
The fertility plasmid or F-plasmid was discovered by Esther Lederberg and encodes information for the biosynthesis of sex pilus to aid in bacterial conjugation. Conjugation involves using the sex pilus to form a bridge between two bacteria cells; this bridge allows the F+ cell to transfer a single-stranded copy of the plasmid so that both cells contain a copy of the plasmid.