Eppendorf AF2200 Plate Reader - Manual (Page 54)

Predefined methods
Eppendorf
® PlateReader AF2200
English (EN)
54
6.2 Method description
6.2.1 Nucleic acid quantification (UV 260 nm microvolume)
For microvolume measurements with the μPlate G0.5 please refer to the dedicated manual.
6.2.2 Nucleic acid quantification (UV 260 nm with factor)
Introduction
The most common technique for measuring nucleic acid concentration is based on measuring the
absorbance at 260 nm. According to the Lambert-Beer law, the amount of absorbed light is proportional to
the concentration of the sample and to the path length of the light passing through a sample.
A = ε * d * c
For the Eppendorf Microplate UV-VIS, 96/F the path lengths of 100, 150, 200, 250 and 300 μL per well have
been determined. Thus, the nucleic acid concentration can easily be calculated.
To assess the purity of the nucleic acid sample, an additional "Ratio" measurement at 280 nm may be
performed to indicate the presence of proteins or organic compounds in the sample. For pure DNA, a 260/
280 ratio between 1.8 - 1.9 is acceptable; for pure RNA, a 260/280 ratio of approximately 2.0 is acceptable.
This method is restricted to the Eppendorf μPlate G0.5.
A Absorbance, OD
ε Extinction Coefficient
d Distance (path length in cm)
c Concentration
Wavelength 260 nm
Sample Type dsDNA ssDNA RNA
average extinction
coefficient ε [μg/mL] 0,02 0,027 0,025
concentration [μg/ml] at
A =1,0 and d = 1,0 50 33 40