Blog article
Contaminants in your nucleic acid sample?
10.07.2024
Measuring absorbance in the UV range has long been an established method for quantifying nucleic acids and determining sample purity. According to the Lambert-Beer law, the degree of light absorption at a 260 nm wavelength correlates directly with the concentration of nucleic acid present in the sample.
Standards factors for nucleic acids are commonly used to calculate sample concentrations when measuring with a standard cuvette with pathlength of 10 mm. For instance, a double-stranded DNA sample exhibiting an absorbance value of 1 at 260 nm corresponds to a concentration of 50 ng/µl, thus the factor is 50. Furthermore, the absorbance ratios measured at various wavelengths are used to evaluate the purity of the sample.
A260/A280
The ratio of absorbance values measured at 260 nm and 280 nm gives an estimation of the purity of a sample and which nucleic acid it contains A lower ratio indicates protein contamination, while a higher ratio suggests the presence of RNA. Pure double-stranded DNA typically shows a ratio of approximately 1.85-1.88, while pure RNA shows a ratio around 2.1.
As a rule of thumb, a ratio of A260/A280 > 1.8 signifies a sample consisting of pure nucleic acid.
A260/A230
Various contaminants may influence the A260/A230 ratio: chaotropic salts used in the extraction, the detergents used in lysis buffers, proteins, and phenol, often used in RNA extraction. The A260/A230 ratio typically ranges from 2.3 to 2.4 for pure double-stranded DNA and from 2.1 to 2.3 for pure RNA.
Low sample concentration
The pathlength used for measuring the absorbance in the NanoDrop is shorter than 10 mm. According to the instrument specifications, it is possible to reliably measure diluted nucleic acid samples down to 2 ng/µl. If you are measuring diluted nucleic acids (<20 ng/µl), it's important to note that the purity ratio should be considered directionally only. This ratio is calculated by dividing very small numerical values, which may introduce variation between replicates.
The effect of the solvent
The pH of the solvent may influence the results. If your DNA or RNA samples are dissolved in water, there could be more variation than with buffered solvents.
Acclaro algorithm and NanoDrop One
The Acclaro Sample Intelligence software can find and correct contamination in your samples and even differentiate DNA from RNA.
How does Acclaro Sample Intelligence work?
The Acclaro Sample Intelligence software operates by analyzing data collected from the NanoDrop One instrument. It uses advanced algorithms as well as built in sensor and image-analysis to detect and correct for potential contamination in samples while also distinguishing between DNA and RNA. By using the absorbance measurements obtained at various wavelengths, the software can accurately assess the purity and integrity of nucleic acid samples and alerts for purity ratio, contaminants, bubbles or broken column. It also provides recommendations for further purification steps if needed, offering valuable guidance to researchers. Overall, the software streamlines the analysis process, ensuring reliable results and facilitating efficient decision-making in nucleic acid research.
Would you be interested in a demo and testing this with your own samples? Contact us!
See our available NanoDrop instruments here.
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