DNA, RNA, and protein strongly absorb ultraviolet light in the 260 to 280 nm range. UV spectroscopy can be used as a quantitative technique to measure nucleic acid concentration and protein contamination. Nucleic acids strongly absorb at 260 nm and less strongly at 280 nm while proteins do the opposite. The general rules for determining the concentrations of nucleic acids at 260 nm are:
- 1 Optical Density (OD) unit of double-stranded DNA is 50 micrograms/ml.
- 1 OD unit of single-stranded DNA is 33 micrograms/ml.
- 1 OD unit of single-stranded RNA is 40 micrograms/ml.
Proteins absorb strongly at 280 nm where 1 OD unit is 1 mg/ml. When using UV spectroscopy for estimating DNA concentrations, it is very important to remove all protein and RNA from the DNA solution. Good estimations can only be made on clean preparations.
An estimate of the purity of a DNA preparation can be made by measuring the absorbance at both 260 nm and 280 nm. Pure solutions of nucleic acid will absorb approximately twice as much at 260 nm as at 280 nm. Experimentally, the ratio of 260 nm/280 nm of a pure DNA solution is between 1.8 to 2.0. As protein contamination increases, the ratio decreases. Additionally, the presence of contaminating oranic solvents, such as phenol, can affect estimations of concentration and purity.
Materials you need are:
Quartz or UV compatible cuvettes
- Fill the cuvette with water or TE buffer. Zero the spectrophotometer at 260 nm with this blank.
- DNA from plasmid and genomic preparations is typically at a concentration exceeding 1 micrograms/microliter. Consequently, DNA is usually diluted before measuring its absorbance. An unfortunate result of this measurement is that the DNA is expended as a result of the dilution. Be sure these is adequate DNA to waste. Start by diluting the DNA sample 1 microliter : 999 microliters of TE buffer (the dilution can be done directly in the cuvette). Mix the dilution thoroughly.
- Measure the optical density (OD). Multiply the resulting OD by 50 micrograms/ml. For a 1:1000 dilution, the mass of DNA is equal to micrograms/microliter.
- Similarly, the same sample can be measured at 280 nm. A ratio of the OD-260nm/OD-280nm is an indicator of DNA purity. A ratio of 1.8 or higher indicates minimal protein contamination.
Measuring DNA concentration is not difficult, is it?