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Index of DNA Purity
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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Update: June 2012
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