Thermo Scientific NanoDrop One, Руководство пользователя

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Learning Center
Acclaro Sample Intelligence
252 NanoDrop One User Guide Thermo Scientific
• original nucleic acid concentration (451.2 ng/μL), which is based on the total
baseline-corrected absorbance (sample plus contaminant) at the analysis wavelength
• corrected nucleic acid concentration (311.9 ng/μL), which is based on the corrected
absorbance (sample minus contaminant) at the analysis wavelength
Theory behind contaminant analysis
UV and UV-visible absorbance measurements are used to quantify nucleic acid and protein
samples at 260 nm and 280 nm, respectively. The analysis is based on the fact that the total
absorbance of a mixture solution at a given wavelength is the sum of the absorbance values of
each component in the mixture.
An ongoing challenge of this method is that a number of materials used in the extraction
process can absorb in various regions across the spectrum. When these contaminants are
present in a sample, they can interfere with the analysis by artificially inflating the absorbance
at the wavelength of interest, which causes the analyte concentration to be overestimated.
Traditionally, purity ratios are used to detect the presence of contaminants that could affect
downstream applications. However, purity ratios do not always provide a complete picture of
possible contamination. When a purity ratio falls outside the expected range, the spectral
profile is often examined qualitatively.
Our Acclaro technology applies a quantitative approach to contaminant analysis. Through
sophisticated mathematical algorithms, Acclaro analyzes the spectral data to identify probable
contaminants in a sample and removes any contribution due to the contaminant from the
sample result. This results in a more accurate concentration value of the analyte of interest and
a more qualitative analysis of the level of contamination.
Since the spectrum of a pure compound is unique to that compound, a mixture spectrum of
mostly known materials that have few interactions can be mathematically broken down into
its component spectra and the components identified. The contaminant analysis algorithm
uses a narrow spectral region (220-285 nm) around the analysis wavelength (260 nm for
nucleic acids, 280 nm for proteins) to determine any absorbance contribution from possible
known contaminants (protein or nucleic acid, and phenol) that absorb in that region. The
entire spectrum is analyzed to determine the presence of other possible contaminants such as
guanidine HCl and/or guanidinium isothiocyanate, which are common reagents used for
nucleic acid purification.
Note Achieving consistent, high quality contaminant analysis results is dependent on the
quality of the measured sample spectra, which is dependent on the maintenance status of
the instrument. For more information, see
Maintenance Schedule .