Using the Spectral Evaluation Function for Quantitative Analysis of Nucleotides and a Simultaneous Pass/Fail Judgment on Nucleotide Sample Purity

User Benefits

- Use the Spectral Evaluation Function to set threshold criteria and provide a pass/fail judgment based on any chosen wavelength or photometric value. - Use the Spectral Evaluation Function to perform quantitative analysis of nucleotides and simultaneously provide a pass/failjudgment on nucleotide sample purity.

Introduction

UV-Vis spectrophotometers are used for quantitative analysis of solution, and in accordance with the Beer-Lambert law, the amount of light absorbed (measured as absorbance) in a cell of fixed length is proportional to the concentration of solution. Nucleic acids are quantified by measuring absorbance at 260nm because light absorption by nucleic acid bases is greatest at this wavelength. However, the amount of light absorbed by nucleic acids varies by base sequence, sequence length, solvent used, and other factors. To account for this variability and obtain accurate quantitative results, an extinction coefficient is typically calculated for each oligonucleotide. The amount of light absorbed by nucleic acids is also affected by the presence of proteins and other contaminating substances that absorb light at a nearby wavelength (proteins absorb light at 280 nm). This Application News uses the UV-1900i Plus UV-Vis spectrophotometer and LabSolutions UV-Vis control software to measure the absorption spectra of nucleic acids and then uses the Spectral Evaluation Function to perform a quantitative analysis using a calibration curve based on absorbances measured at the wavelengths of maximum absorption. The Spectral Evaluation Function was also used to simultaneously check for contaminating substances that affect the quantitative analysis by providing a pass/fail judgment on nucleic acid sample purity based on the ratio of absorbance at 260 nm and 280 nm (OD260/OD280).

February 25, 2025 GMT