Thermo Scientific NanoDrop One, User Manual

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4
Measure Microarray
Thermo Scientific NanoDrop One User Guide 33
Calculated nucleic acid concentrations are based on the
absorbance value at 260 nm, the factor used and the
sample pathlength. A single-point baseline correction (or
analysis correction) may also be applied.
Concentration is reported in mass units. Calculators are
available on the Internet to convert concentration from
mass to molar units based on sample sequence.
Absorbance values at 260 nm, 280 nm and sometimes
230 nm are used to calculate purity ratios for the
measured nucleic acid samples. Purity ratios are
sensitive to the presence of contaminants in the sample,
such as residual solvents and reagents typically used
during sample purification.
Measured Values
A260 absorbance
Note : The absorbance value at 750 nm is subtracted from all wavelengths
in the spectrum. As a result, the absorbance at 750 nm is zero in the
displayed spectra. Also, for micro-volume absorbance measurements and
measurements taken with nonstandard (other than 10 mm) cuvettes, the
spectra are normalized to a 10 mm pathlength equivalent.
• Nucleic acid absorbance values for all Microarray sample types are
measured at 260 nm using the 750-corrected and normalized
spectrum.
• If Analysis Correction is selected, the absorbance value at the
correction wavelength is subtracted from the absorbance at 260 nm.
• If one or more dyes are selected, the dye correction values at 260 nm
are also subtracted from the absorbance at 260 nm.
• The final corrected absorbance at 260 nm is reported and used to
calculate sample concentration.
A280 absorbance
• 750-corrected and normalized absorbance value at 280 nm (minus the
A280 dye correction) is used to calculate an A260/A280 ratio.
Dye concentrations are calculated from the absorbance
value at the dye’s analysis wavelength, the dye’s
extinction coefficient, and the sample pathlength. A
sloped-line dye correction may also be used. Dye absorbance
• Dye absorbance values are measured at specific wavelengths. See
Dye/Chromophore Editor for analysis wavelengths used.
• If Sloping Dye Correction is selected, a linear baseline is drawn
between 400 nm and 750 nm and, for each dye, the absorbance value
of the sloping baseline is subtracted from the absorbance value at each
dye’s analysis wavelength. Baseline-corrected dye absorbance values are
reported and used to calculate dye concentrations.
Dye correction
• Pre-defined dyes have known correction values for A260 and A280.
See Dye/Chromophore Editor for correction values used.
• A260 dye corrections are subtracted from the A260 absorbance value
used to calculate nucleic acid concentration, and from the A260
absorbance value used to calculate the
A260/A280 purity ratio .