Extraction and Analysis of Plasmid Dna from E. Coli Cells

Topics: Molecular biology, DNA, Restriction enzyme Pages: 9 (2774 words) Published: March 7, 2013
Extraction and Analysis of Plasmid DNA from E. coli Cells
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 uses the fact that cell membranes can be lysed under extreme heat. Ethanol-salt precipitation was performed to pellet the plasmid DNA following removal of excess RNA and protein components. After extracting the unknown plasmid from E.coli, restriction enzymes were used to create a restriction map of the recognition domains for different enzymes…. The purpose of this study was to isolate and characterize an unknown plasmid within Escherichia coli, strain JM109, and qualitatively assess the purity of the extract by comparison to a standard curve. Following isolation of the plasmid, single and double restriction enzyme digests were performed in order to create a restriction map of recognition domain locations for restriction enzymes on the unknown plasmid.

Materials and Methods
Escherichia coli JM109, containing an unknown plasmid, was obtained from New England Biolabs. Absorbance values at 260 nm, and 280 nm, for determination of plasmid purity, were obtained using a Cary Varian 50 Bio UV spectrophotometer. 2-Log DNA reference ladder from New England Biolabs was used as a comparison of the band migration distances of plasmid samples. All plasmid samples were prepared with Bromophenol Blue loading dye. Restriction enzyme digestion samples also contained 10 X NEB enzyme buffers 4 obtained from New England Biolabs.

Plasmid Purification
LB media containing 50 µg/mL ampicillin was inoculated with E.coli and grown overnight at 37˚C with shaking. 1.5 mL of bacterial culture was centrifuged for 20 seconds at maximum speed to pellet bacteria. The supernatant was poured out and any excess was tapped out onto a paper towel. The pellet was then re-suspended in 500 µL of DNase-free water, mixed, centrifuged again, and the supernatant was removed. The pellet was then re-suspended in 350 uL of STET solution (8% (w/v) sucrose, 50 mM Tris buffer (pH 8.0), 50 mM EDTA, and 5% (v,v) Triton X-100, with 0.3 mg/mL lysozymes and 0.3 mg/mL of bovine pancreatic ribonuclease...
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