RNA bands appear faint and smeary and are only detected in amounts ≥25–30 ng (0.5:1 RNA:DNA ratio). RNA contamination can sometimes be detected by agarose gel analysis with routine ethidium bromide staining, although not quantified effectively. Spectrophotometric measurements do not differentiate between DNA and RNA, so RNA contamination can lead to overestimation of DNA concentration. RNA may inhibit some downstream applications, but it will not inhibit PCR. In contrast, A 260/A 280 ratios measured in a low-salt buffer with slightly alkaline pH are generally reproducible.Įffect of RNA contamination on spectrophotometric readingsĭepending on the DNA isolation method used, RNA will be co-purified with genomic DNA. A 260/A 280 ratios measured in water also give rise to a high variability between readings (see figure Effect of solvent on A 260/A 280 ratio) and the ratios obtained are typically <1.8, resulting in reduced sensitivity to protein contamination (7). This is most likely due to differences in the pH of the water caused by the solvation of CO2 from air. A 260 values are reproducible when using low-salt buffer, but not when using water. Phenol contamination mimics both higher yields and higher purity, because of an upward shift in the A 260 value.Įffects of solvents on spectrophotometric readingsĪbsorption of nucleic acids depends on the solvent used to dissolve the nucleic acid (7). Tip: Phenol has an absorbance maximum of 270–275 nm, which is close to that of DNA. Tip: Spectrophotometric measurements do not differentiate between DNA and RNA, so RNA contamination can lead to overestimation of DNA concentration. Tip: If you use more than one cuvette to measure multiple samples, the cuvettes must be matched. When working with small amounts of DNA, such as purified PCR products or DNA fragments extracted from agarose gels, quantification via agarose gel analysis may be more effective (see Agarose gel). An example of the calculation involved in nucleic acid quantification when using a spectrophotometer (see Spectrophotometric measurement of DNA concentration). This relation is valid only for measurements made at neutral pH, therefore, samples should be diluted in a low-salt buffer with neutral pH (e.g., Tris♼l, pH 7.0). An absorbance of 1 unit at 260 nm corresponds to 50 µg genomic DNA per ml (A 260 =1 for 50 µg/ml based on a standard 1 cm path length. For greatest accuracy, readings should be between 0.1 and 1.0. DNA concentration can be determined by measuring the absorbance at 260 nm (A 260) in a spectrophotometer using a quartz cuvette.
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