Plasmid DNA may appear in several conformations.
- Covalently closed circular DNA (cccDNA) – both strands of the plasmids DNA are intact. (Supercoiled DNA, Relaxed circular DNA)
- Open circular DNA – One nucleotide stand is broken
Super coiled DNA is move faster than open circular and genomic DNA because it compact conformation.
Plasmid DNA extraction methods.
- Separation basis on size : Usually a combination of enzymes and chemical reagents (Lysozyme, EDTA, Sucrose, SDS) cells are gently lysed under specific conditions then the breakage of chromosomal DNA is minimized. Most of the genomic DNA is trapped in the cell debris, plasmid are extracted by centrifugation.
- Alkaline Lysis : Culture media is exposed to the alkaline solution (SDS,NaOH) in alkaline conditions plasmids chromosomal DNA and proteins get denatured. in the rapid neuralizations with high salt buffer (potassium acetate) Plasmids re-anneal; gDNA and proteins are not.
- Boiling Lysis Method : cells are partially lysed, allowing plasmid molecules to escape
- Ethidium Bromide- CsCl Density Gradient Centrifugation: In agarose gel electrophoresis, a dye called ethidium bromide is widely applied to see the DNA. When DNA is treated with ethidium bromide and examined under UV light, the DNA fluoresces, enable to detect the DNA bands. A density gradient is created by the salt CsCl dissolved in water. After centrifuging, the nucleic acid sample is placed on top of the CsCl gradient. The thicker nucleic acids move along the gradient and create bands (used to separate non-supercoiled DNA from supercoiled DNA molecules)
References
- De Ungria, M. C. A., Tillett, D., Neilan, B. A., Cox, P. T., & Lee, A. (1998, December). A Novel Method of Extracting Plasmid DNA fromHelicobacterSpecies. Helicobacter, 3(4), 269–277. https://doi.org/10.1111/j.1523-5378.1997.06085.pp.x-i1
- Agarose gel electrophoresis. (n.d.). Wikipedia. https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis
- Garger, S., Griffith, O., & Grill, L. (1983, December). Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient. Biochemical and Biophysical Research Communications, 117(3), 835–842. https://doi.org/10.1016/0006-291x(83)91672-8