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The most common method is alkaline lysis, which involves the use of a high concentration of a basic solution, such as sodium hydroxide, to lyse the bacterial cells. [15] [16] [17] When bacteria are lysed under alkaline conditions (pH 12.0–12.5) both chromosomal DNA and protein are denatured; the plasmid DNA however, remains stable.
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.
In this example, a gene from mammalian gene library will be subcloned into a bacterial plasmid (destination platform). The bacterial plasmid is a piece of circular DNA which contains regulatory elements allowing for the bacteria to produce a gene product (gene expression) if it is placed in the correct place in the plasmid. The production site ...
The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. [1] DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and ...
Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication, and the resulting recombinant DNA is introduced into the organism from which the replication sequences were obtained ...
A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. [ 1 ] [ 2 ] [ 3 ] F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division.
The term plasmid was coined in 1952 by the American molecular biologist Joshua Lederberg to refer to "any extrachromosomal hereditary determinant." [11] [12] 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 ...
AT-rich region: important for DNA strand opening and is located adjacent to the inverted repeat sequences. [1] [3] [5] [8] [11] [12] The oriT is a noncoding region of the bacterial DNA. [13] Due to its important role in initiating bacterial conjugation, the oriT is both an enzymatic substrate and recognition site for the relaxase proteins.
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