Thermo Scientific NanoDrop One User Manual page 33

For the dsDNA, ssDNA and RNA
applications, the generally accepted factors
for nucleic acids are used in conjunction
with Beer's Law to calculate sample
concentration. For the Custom Factor
application, the user-specified factor is
used.
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.
Thermo Scientific
Factors Used
• dsDNA (factor = 50 ng-cm/µL)
• ssDNA (factor = 33 ng-cm/µL)
• RNA (factor = 40 ng-cm/µL)
• Custom Factor (user entered factor between
15 ng-cm/µL and 150 ng-cm/µL
Measured Values
Note: 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.
A260 absorbance
• Nucleic acid absorbance values are measured at 260 nm
using the normalized spectrum. This is the reported A260
value if Baseline Correction is not selected.
• If Baseline Correction
at the correction wavelength is subtracted from the
absorbance at 260 nm. The corrected absorbance at
260 nm is reported and used to calculate nucleic acid
concentration.
A230 and A280 absorbance
• Normalized and baseline-corrected (if selected)
absorbance values at 230 nm and 280 nm are used to
calculate A260/A230 and A260/A280 ratios.
Sample Pathlength
• For micro-volume measurements, the software selects
the optimal pathlength (between 1.0 mm and 0.03 mm)
based on sample absorbance at the analysis wavelength.
• For cuvette measurements, pathlength is determined by
the cuvette Pathlength setting in the software (see
General Settings).
• Displayed spectra and absorbance values are normalized
to a 10 mm pathlength equivalent.
4
Nucleic Acid Applications
Measure dsDNA, ssDNA or RNA
is selected, the absorbance value
NanoDrop One User Guide 31