UV Spectrophotometry using absorbance at 260 nm
Absorbance has been the method of choice for routineand RNA for decades. It is simple and convenient to use as there is no further sample treatment (other than DNA extraction) or reaction with other substances are required. However, it is not very specific (it measures all nucleic acids as a whole) and sensitive to contaminants, so it demands very pure DNA to be accurate. , how to measure it and how it affects quantification.
Absorbance of DNA samples at 260 nm is currently most often measured using abut it is also possible to use a cuvette spectrophotometer or a .
In this method the concentration of a substance is calculated according to the Beer-Lambert law based on its absorbance. The following formula can be obtained from the original formulation of the law:
Where A = absorbance at a given wavelength, ε = extinction coefficient, b = pathlength of the spectrophotometer, c = concentration of the sample.
So, for a pathlength of 1 cm, the concentration is equal to the absorbance at 260 nm (the absorption peak of nucleic acids), divided by the extinction coefficient.
Formula to calculate DNA concentration
dsDNA has an extinction coefficient of 0.02 (µg/mL)-1 cm-1:
The same formula can be used with the respective extinction coefficients for ssDNA (absorbance x 37 µg/mL) and ssRNA (absorbance x 40 µg/mL). It is important to note that the formula is only valid for large nucleic acid molecules with a similar proportion of all nucleotides, such as genomic DNA and plasmids. For oligonucleotides and other short nucleic acid molecules such as miRNAs, the extinction coefficient must be calculated from the sequence of the oligonucleotide.
The diphenylamine method (Dische's Test)
Another method of quantifying DNA by absorbance is the diphenylamine method which is based on the reaction of diphenylamine with deoxyribose sugar to form a blue complex. This method is time-consuming, has a low sensitivity and is therefore no longer used in most laboratories. The measurement is made at 595 nm with a standard.