Quantification of DNA

Using spectrophotometry, the optical density (OD) of light absorbance by DNA molecules in a known water concentration can be used to determine DNA concentration and yield. An aliquot of DNA is taken to measure the light absorbance at 230, 260, and 280 nm wavelengths. Some methods of DNA quantitation involve fluorescent dyes that intercalate between the double helix. The latter is subjectively more accurate but may be prone to errors in making the dye solution. DNA can be quantified mathematically by considering the OD of light in a known aliquot.

• DNA is detectable at wavelengths of 260 nm.
• Certain salts, phenols, and carbs are detectable around 230 nm wavelengths.
• Protein and RNA can show absorbance in the 280 nm wavelength range.
• The 260 (DNA) absorbance is compared to 230 and 280 to generate a ratio.
• A good ratio of DNA (260:280) and (260:230) for optimal purity of DNA in a sample is approximately 1.8.
• High ratios (>1.8 may indicate RNA contamination) and low ratios (<1.8 may indicate contamination from protein or phenols).
• Labs are responsible for determining what is acceptable for their own ratio cutoffs and poor ratio samples typically need to be cleaned and concentrated from a column to remove impurities.