Extraction and Analysis of Plasmid Dna from E. Coli Cells
Extraction and Analysis of Plasmid DNA from E. coli Cells
Introduction
A plasmid is an extra-chromosomal element, often a circular DNA. Since a plasmid is by definition an extra-chromosomal element, it cannot make use of any origin of DNA replication in a chromosome (BP site). Meaning that DNA synthesis within a plasmid depends on having an origin of DNA synthesis of its own. Plasmids are often found in bacterial cells, in which they are used as transfer agents for transmitting various antibiotic resistance and stress response genes (site). Gene transfer between bacteria via a bacterial plasmid allows organisms to transfer genetic information horizontally, rather than being limiting to passing it between generations. Bacterial cell’s ability to adapt to their environment and reproduce so quickly has proven useful to obtain large quantities of particular plasmid. Biologically useful plasmids are often synthesized and inserted into strains of bacteria, which reproduce them at an elevated rate. Plasmid replication through bacterial reproduction provides a cost effective alternative to preparing plasmids using other methods (site).
In order to isolate plasmid DNA from a bacterial host, the cell membrane must first be destroyed to release the contents of the cell (source). Typical methods use lysozymes to break open the membrane. The result of cell lysis is the release of a single loop of genomic DNA and small chunks of circular plasmid DNA. The plasmid DNA can be isolated genomic DNA and RNA impurities by a series of extractions, using RNase lysozymes and isopropanol to remove excess RNA and proteins from the cell (source).
EDTA is added to the buffer solutions to inhibit endonuclease enzymes by complexing free Mg2+, a common cofactor for many DNA binding enzymes (source). Triton X100 is also added as a detergent for dissolving the lipid components of E.coli without disrupting the enzymes. Boiling lysis is a cheap alternative to enzymatic cell lysis that
Introduction
A plasmid is an extra-chromosomal element, often a circular DNA. Since a plasmid is by definition an extra-chromosomal element, it cannot make use of any origin of DNA replication in a chromosome (BP site). Meaning that DNA synthesis within a plasmid depends on having an origin of DNA synthesis of its own. Plasmids are often found in bacterial cells, in which they are used as transfer agents for transmitting various antibiotic resistance and stress response genes (site). Gene transfer between bacteria via a bacterial plasmid allows organisms to transfer genetic information horizontally, rather than being limiting to passing it between generations. Bacterial cell’s ability to adapt to their environment and reproduce so quickly has proven useful to obtain large quantities of particular plasmid. Biologically useful plasmids are often synthesized and inserted into strains of bacteria, which reproduce them at an elevated rate. Plasmid replication through bacterial reproduction provides a cost effective alternative to preparing plasmids using other methods (site).
In order to isolate plasmid DNA from a bacterial host, the cell membrane must first be destroyed to release the contents of the cell (source). Typical methods use lysozymes to break open the membrane. The result of cell lysis is the release of a single loop of genomic DNA and small chunks of circular plasmid DNA. The plasmid DNA can be isolated genomic DNA and RNA impurities by a series of extractions, using RNase lysozymes and isopropanol to remove excess RNA and proteins from the cell (source).
EDTA is added to the buffer solutions to inhibit endonuclease enzymes by complexing free Mg2+, a common cofactor for many DNA binding enzymes (source). Triton X100 is also added as a detergent for dissolving the lipid components of E.coli without disrupting the enzymes. Boiling lysis is a cheap alternative to enzymatic cell lysis that
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