In order to measure DNA content you can use UV Spectrophotometer with the advantages are nondestructive and allows the sample to be recovered for further analysis or manipulation. Spectrophotometry uses the fact that there is a relationship between the absorption of ultraviolet light by DNA/RNA and its concentration in a sample. In this particular posting, We try to share with you facts about the relationship between DNA/RNA with the wavelength in the Spectrophotometer assay.
- The absorption maximum of DNA/RNA is approx 260 nm. This figure is an average of the absorption of the individual nucleotides that vary between 256 and 281 nm.
- In the case of RNA, the concentration of a sample containing RNA may be calculated following Equation:
And this equation for DNA concentration:
The equation above describe that when the OD-260 of the sample is 1 the concentration of RNA will be approx 40 micrograms/mL (50 micrograms/mL for DNA).
- We can also assess the degree of purity of the nucleic acids by examining the absorption at other wavelengths in which protein and polysaccharides have known absorption maxima. Proteins are known to absorb strongly at 280 nm and polysaccharides may be identified by their maximum at 230 nm.
- Therefore, in assessing the purity degree of the nucleic acid sample we use the ratio of measurements of these three wavelengths 230 nm, 260 nm, and 280 nm.
- For example a sample containing only RNA following an extraction method is judged as being uncontaminated if the ratio is 1 :2 : 1, and for DNA is 1 : 1.8 : 1 (it reflects OD-230 : 260 : 280 ratio). If there is significant deviation from the ratio, then it is evident that contaminants are present and that further purification of the sample is necessary.
In many cases, the purity and the concentration may be further obscured by the presence of reagents that are used in the extraction process itself. Some of these have characteristics that are evident on a spectrophotometric scan that includes the three wavelengths indicated. Therefore, when using spectrophotometry in the analysis of DNA or RNA it is necessary to be aware of the potential problems that may result in misleading ratio. Further, when analyzing ratios and concentrations of DNA or RNA spectrophotometrically it is also necessary not only to derive readings at 280,260, and 230 nm but also to scan throughout the range 200-320 nm. Trace amounts of reagents used in the extraction process can influence adversely and provide misleading data that may affect any subsequent manipulation.