How is UV Vis spectroscopy used for DNA analysis?
Quality and quantity determination of a DNA sample can be crucial for the success of downstream analysis and, in most molecular biology labs, is typically carried out by UV Vis spectroscopy. For DNA analysis, it is typically advised to scan the complete spectral range from 230 to 320 nm. Within this range, the wavelengths of most interest are 260, 280, 230 and 320 nm, respectively.
With this application note, you will learn how to analyze DNA samples using METTLER TOLEDO's UV5Nano - a Microvolume UV Vis Spectrophotometer, which requires only 1-2 µL of sample and delivers result in seconds.
DNA analysis at 260 nm
DNA has its absorbance maximum at 260 nm. When measured in a 10 mm cell, the concentration of double-stranded DNA is approximated as 50 µg/mL per 1 absorbance unit, using the average extinction coefficient of 0.020 (µg/mL)-1 cm-1. When taking a reading at 260 nm, the presence of contaminants, which also absorb at 260 nm, can lead to an overestimation of DNA content. Therefore, a background correction at 320 nm is often applied.
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Ratio 260/280 and 260/230
The absorbance ratio 260/280 is a good indicator of protein contamination: when ≥ 1.8, it indicates a pure DNA sample.
The absorbance ratio 260/230, when smaller than 1.8, indicates contamination probably caused by organic compounds or chaotropic agents, which absorb at 230 nm.
