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4
Nucleic Acid Applications
Measure Oligo DNA or Oligo RNA
Calculations for Oligo DNA and Oligo RNA Measurements
As with the other nucleic acid applications, the
Oligo applications use the
equation
to correlate absorbance with
concentration based on the sample's extinction
coefficient and pathlength. Because
oligonucleotides are short, single-stranded
molecules (or longer molecules of repeating
sequences), their spectrum and extinction
coefficient (
) are closely dependent on base
composition and sequence.
(The generally accepted extinction coefficients
and factors for single-stranded DNA and RNA
provide a reasonable estimate for natural,
essentially randomized, sequences but not for
short, synthetic oligo sequences.) To ensure the
most accurate results, we use the exact value of
to calculate oligonucleotide concentration.
260
The NanoDrop software allows you to specify
the base sequence of an oligonucleotide before
it is measured. For any entered base sequence,
the software uses the equation at the right to
calculate the extinction coefficient.
Tip: The extinction coefficient is wavelength
specific for each oligonucleotide and can be
affected by buffer type, ionic strength and pH.
58
NanoDrop One User Guide
(550 AU * 55 ng-cm/µL) = 30,250 ng/µL
Note For measurements with 10 mm pathlength cuvettes, the upper
absorbance limit is 1.5 AU, which is approximately 75 ng/µL for dsDNA.
Related Topics
•
Detection Limits for All Applications
Beer-Lambert
Extinction Coefficients for Oligonucleotides
The software uses the nearest neighbor method and the
following formula to calculate molar extinction
coefficients for specific oligonucleotide base sequences:
N 1
–
260
=
1
where:
= molar extinction coefficient in L/mole-cm
1 =
nearest neighbor
2 =
individual bases
3 =
modifications, such as fluorescent dyes
N 1
–
N
1
2
3
–
+
2
1
Thermo Scientific
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