NanoPhotometer
®
P-Class
User Manual
Version 2.1
Page 14 / 70
Pure Nucleic Acid Poly dAdT
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
210.0
260.0
310.0
360.0
410.0
Wavelength (nm)
A
b
s
o
rb
a
n
c
e
(
A
)
Wave = 260.0 Abs = 0.567
Wave = 280.0 Abs = 0.409
Pure Nucleic Acid Poly dAdT
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
210.0
260.0
310.0
360.0
410.0
Wavelength (nm)
A
b
s
o
rb
a
n
c
e
(
A
)
Wave = 260.0 Abs = 0.567
Wave = 280.0 Abs = 0.409
different ratios due to variations in wavelength accuracy. But each instrument will give consistent results within
itself.
Concentration also affects 260/280 readings. If a solution is too dilute, the readings will be at the instrument’s
detection limit, and results may vary as there is less distinction of the 260 peak and the 280 slope from the
background absorbance. This is one reason why the Abs 260 value by using the submicroliter cell (NanoVolume
applications) should stay between than 0.01 and 1.50 for accurate measurements.
An elevated absorbance at 230 nm can indicate the presence of impurities as well; 230 nm is near the absorbance
maximum of peptide bonds and also indicates buffer contamination since TRIS, EDTA and other buffer salts absorb
at this wavelength. When measuring RNA samples, the 260/230 ratio should be > 2.0; a ratio lower than this is
generally indicative of contamination with guanidinium thiocyanate, a reagent commonly used in RNA purification
and which absorbs over the 230 - 260 nm range. A wavelength scan of the nucleic acid is particularly useful for
RNA samples.
The instrument can display 260/280 and 260/230 ratios.
Fluorescent dye incorporation
To determine the dye incorporation rate, the absorbance reading at the wavelength reported for maximum
absorbance of the fluorescence dye is used. The corresponding extinction coefficient of the dye is used in the
Lambert-Beer Law to determine the dye concentration (c = A / (e * d)). Comparing these values with the DNA
concentration gives a dye incorporation rate. For further details please refer to 12.2 Nucleic acid fluorescent dye
incorporation.
Use of Background Correction
Background correction at a wavelength totally separate from the nucleic acid and protein peaks at 260 and 280
nm, respectively, is sometimes used to compensate for the effects of background absorbance. The wavelength
used is 320 nm and it can allow for the effects of turbidity, high absorbance buffer solution and the use of reduced
aperture cells.
If it is used, there will be different results from those when unused, because Abs 320 is subtracted from Abs 260
and Abs 280 prior to use in equations:
Concentration = (Abs 260 - Abs 320) * Factor
Abs ratio = (Abs 260 - Abs 320) / (Abs 280 - Abs 320)
Abs ratio = (Abs 260 - Abs 320) / (Abs 230 - Abs 320)
If your laboratory has not used background correction before, set this option to OFF.
The use of background correction can remove variability due to handling effects of low volume disposable cells.
Spectral scan of nucleic acid
Note:
absorbance maximum near 260 nm and absorbance minimum near 230 nm
flat peak near 260 nm and steep slope at 280 nm
very little absorbance at 320 nm
Operation of the instrument for Nucleic Acid measurements is described in the following sections.
DNA and RNA are very similar, whilst in Oligo it is possible to calculate the factor from the composite bases by entering
the proportions of the 4 bases.