DNA Purity Calculator | Assess Nucleic Acid Sample Quality

Whether a sample with non-ideal purity ratios remains usable depends on your application's sensitivity to contaminants. Generally: (1) For standard PCR with robust targets, samples with A260/A280 ratios of 1.6-2.1 often work adequately. (2) For qPCR, a wider range of 1.7-2.1 can be acceptable, but consistent purity is important for comparative studies. (3) For applications like high-throughput sequencing, cloning, or enzymatic digestions, staying close to ideal ratios (1.8-2.0 for DNA) becomes more critical. (4) For RNA work, especially with sensitive applications like microarrays or RNA-Seq, stricter adherence to ideal ratios is necessary. Always validate questionable samples in your specific application before proceeding with valuable or irreplaceable specimens.

A260/A230 ratios typically show greater variability than A260/A280 ratios for several reasons: (1) Lower absorbance at 230nm makes these measurements more susceptible to background noise and instrument limitations. (2) Many common laboratory contaminants absorb at 230nm, including buffer components, guanidine salts, phenol, carbohydrates, and even different elution buffers. (3) pH significantly affects 230nm readings—small changes in sample pH can cause large fluctuations in A260/A230 ratios. (4) The measurement at 230nm is more sensitive to cuvette positioning errors. For more consistent A260/A230 readings, ensure background subtraction using the same buffer as your sample, standardize the pH of your solutions, and use high-quality quartz cuvettes with consistent orientation.

pH significantly impacts nucleic acid absorbance readings and can affect purity ratio interpretation. Higher pH increases absorbance at 260nm while having minimal effect at 280nm, artificially increasing the A260/A280 ratio. For example, the same DNA sample might show an A260/A280 ratio of 1.6 when dissolved in acidic water (pH 5.5) but 1.8-1.9 when in a basic solution like TE buffer (pH 8.0). This explains why seemingly contradictory results can occur when measuring the same sample in different buffers. For consistent and comparable measurements: (1) Standardize the buffer used for all samples, (2) Calibrate your spectrophotometer with the same buffer used for samples, (3) Ideally use a buffer with pH ~8.0 for DNA and RNA measurements, and (4) Report both the ratio and the buffer/pH when documenting results.