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Name: Sara G.
Status: Student
Age: 18
Location: N/A
Country: N/A
Date: December 2002


Question:
why is the ratio 260/280 used as an index of DNA purity?



Replies:
When DNA is isolated from organisms, frequently there remains protein present in the DNA solution; protein is tightly bound to DNA and complete removal of protein is not always possible. To determine the concentration and purity of the DNA solution, the absorbance of UV light is measured in a spectrophotometer. Both protein and DNA absorb UV light, but they have different absorbance curves. The peak of light absorption is at 260 nm for DNA and at 280 nm for protein. When you would run a spectrum of absorbance with varying wave length, you would see that both curves slightly overlap in the area between, and including, 260 and 280 nm.

Thus, when a solution contains both protein and DNA, absorbance at 260 nm is mainly due to the DNA present, but a little bit by the protein. At 280 it is the other way round. By dividing the two absorbance values, one can calculate the purity of the DNA solution. Is the solution relatively free of protein, then one can take the absorbance at 260 nm as a measure for concentration of DNA.

Trudy Wassenaar


Strictly speaking, the ratio is not "260/280" per se, rather the ratio is the absorbance of 260 nm wavelength (from the light spectrum) to the absorbance of 280 nm wavelength. The first is the wavelength that is in the center of the bell curve of absorbance for nucleic acid, and the second is the wavelength that is in the center of the bell curve of absorbance for proteins. Accordingly, a pure sample of DNA, substantially free of protein, will have a dramatically high 260/280 ratio, in that the absorbance at 280 will be a small number. In contrast, a DNA preparation that is heavily contaminated with protein will have a lesser 260/280 ratio.

Now, as to why 260/280 is used as an index of DNA purity, it is due to the ease of the assay: merely take a known quantity of a DNA preparation, dilute in double distilled water to a determined extent, place an aliquot of the solution into a suitable cuvette, and measure the absorbances at 260 nm and 280 nm in a light spectrometer, do the division, and you are done.

Donald Silvert



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