Purpose
This Reference Material can be used for quality control and assurance of nucleic acid purity measurements using the 260/280 nm ratio method.
Description and Discussion
The ratio of the UV absorbance values at 260 nm and 280 nm is an indication of the purity of nucleic acid preparations.Pure DNA and RNA solutions will have ratios of ≥ 1.8 and ≥2.0, respectively. One potential problem with this method is that the 280 nm measurement is taken on a steep slope in the absorbance spectrum, so that a small variation in wavelength at 280 nm will have a large effect on the measured absorbance and hence the 260/280 ratio. It is important to note that avariation of ±1 nm, i.e. within the wavelength specificationtolerance of many spectrophotometers, can give rise to asignificant difference in the 260/280 ratio.
Instrument qualification using Certified Reference Materials is an essential part of any quality control programme. Reliablereference materials using DNA solutions are difficult toproduce, are inherently unstable, and the materials themselvesare often difficult to characterize, handle, package, store and certify. The DNACON reference from Starna was developed as a reliable, stable and NIST traceable reference material for thevalidation of this method. The reference consists of a solution of a synthetic material whose spectral characteristics closely mimic those of DNA, permanently heat-sealed in a far-UVquartz cell. By replicating an actual DNA sample, DNACONgives a reliable direct indication of the quality of the overall measurement process.
Typical spectra
Absorbance values are certified at 260 nm, 280 nm and 330 nm for spectral bandwidths of 1.0, 2.0, 3.0, 4.0 and 5.0 nm . The value at 330 nm is used as a “background” correction, as in the normal methodology. From these values a corrected 260/280 ratio is calculated and reported for each bandwidth using the formula:
Ratio = (A260– A330)
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(A280 – A330)
The typical calculated ratio is 1.9