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Role of ethidium bromide in CTAB method?

Updated: 8/21/2019
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Q: Role of ethidium bromide in CTAB method?
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What is the role of Ethidium bromide in plasmid isolation?

Ethidium bromide is an intercalator, meaning it inserts itself between the base pairs of DNA. Linear DNA pieces like the genomic DNA fragments bind more ethidium bromide than the circular plasmid DNA. The solution is placed into a tube that is spun extremely fast (roughly 50,000 revolutions per minute) in an ultracentrifuge for about a day. During this time the cesium chloride forms a gradient of lower density at the top of the tube and higher density at the bottom. The genomic and plasmid DNA form tight bands in this gradient. Since the plasmid DNA binds less ethidium bromide it is more dense and is located lower in the tube than the genomic DNA. RNA forms a separate band at the bottom of the tube. These three bands can be visualized by UV light.


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What is the role of Cscl and ethidium bromide in plasmid purification?

The CsCl forms a gradient and the molecules migrate according to their density until they float at their individual isopycnic points (the point in the gradient that equals the buoyant density of the molecule). However, plasmid DNA and contaminating chromosome have about the same density and cannot be separated easily. This is rectified, however, by the addition of ethidium bromide. Density is a function of AT/GC ratio, but it is also a function of conformation. For supercoiled DNA, there is more DNA per unit volume than for relaxed DNA. Intercalation of ethidium bromide into DNA causes the helix to unwind (negative supercoiling) and become more relaxed. However, negative supercoiling only relaxes the DNA to a point. Further unwinding induces supercoiling in the opposite direction. When the DNA is circular and the ends are connected, the plasmid "kinks up" into a very tight knot. Thus, ethidium bromide causes the plasmid density to be increased.


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