UV/VIS of nucleic acids and proteins
The optical density of biomolecules such as DNA, RNA and proteins is influenced by pH, temperature and salt concentration (1). The absorbance of a solution decreases with increasing ionic strength, e.g. DNA has approximately 23% lower absorbance when dissolved in buffered saline rather than water (2). Similarly, exact molar extinction coefficients are dependent upon the nucleotide sequence for nucleic acids or the amino acid type and amino acid sequence in peptides or proteins. Therefore molar extinction coefficients αλ are not commonly applied in molecular biology to determine biomolecular concentrations. Instead scientists prefer to use empirically determined standard coefficients. Because these standard coefficients are derived from optical density measurements at 1 cm, they are only valid if the pathlength used for the sample measurement is also 1 cm. For instance, if the optical density of a double stranded DNA sample with 50 μg DNA/ml water is equal to one absorbance unit at 1 cm pathlength, then the standard coefficient of double stranded DNA is 50 μg/ml, also at 1 cm pathlength.
The use of standard coefficients leads to approximations rather than precise determinations of biomolecule concentration within a sample. However standard coefficients have proven to be easy to use with sufficient accuracy for daily laboratory work. To avoid large divergences assay conditions should be considered when determining concentration of nucleic acids or proteins on the basis of standard coefficients.
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Microplates for the UV/VIS determination of nucleic acids at 260 nm and proteins at 280 nm
Standard microplates are only partially suitable for transmission in the UV range, as polystyrene absorbs UV light especially in the short-wavelength range below 400 nm. Greiner Bio-One μClear®; Microplates with a thin polystyrene based film that display much lower background values and can be used down to 340 nm. The adaptation of the patented μClear®; process technology to a new, innovative UV-transparent material enabled the production of microplates that extend the transmission range down to 230 nm.
For the determination of nucleic acid and protein concentrations at 260 nm without background interference, UV-Star®; Microplates are the ideal alternative to expensive and fragile quartz glass plates or cuvettes.
UV-Star®; Microplates are DMSO resistant, can be stored at -20 °C without issue, and are available in 96 well, 96 well half area, and 384 well formats.
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1.) Willfinger, W. W. 1997. Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid Purity. NBioTechniques 22:474-481. 2.) Mc Gown, E. L. 2000. UV Absorbance Measurements of DNA in Microplates. BioTechniques 28:60-64.
