DNA/RNA Quantification Using a 2mm Cuvette and a Tidas Spectrometer
Concentrations of DNA in solution (31µg/mL and 561µg/mL) were measured with a spectrometer and UV/Vis light source in a cuvette. A 2mm pathlength cuvette does not require a pre-measurement dilution within this concentration range, thus a potential source of error was eliminated. Although a 2mm cuvette has a total internal volume of 0.7mL, only 350µL is required to obtain an accurate measurement.
Standard solutions of DNA (Sigma D1626) were prepared gravimetrically using 18.2MΩ/cm ultrapurified water as a solvent. Solutions were prepared between 0.0µg/mL and 561.1µg/mL.
Measurements were taken in triplicate using a 2mm cuvette (WPI #CUV2102-1) in a standard cuvette holder. An appropriate cuvette spacer (WPI #89342)allowed for consistent placement of the cuvette within the holder. The cuvette holder was attached via two 500µm SMA-terminated fiber optic cables to a UV/Vis light source (WPI #D4H).
Data were collected in 1nm increments across the full range of the instrument (190nm-720nm). The instrument was configured such that reference measurements yielded an 80% total intensity. All measurements utilized 18.2MΩ/cm ultrapurified water as a reference solution.
Experimental results are presented in Table 1:
||Absorbance @260nm [AU]
|0.00||0.002 ± 0.001|
|30.83||0.101 ± 0.009|
|56.59||0.200 ± 0.004|
|85.49||0.311 ± 0.005|
|115.66||0.409 ± 0.003|
|143.94||0.497 ± 0.009|
|281.63||0.879 ± 0.010|
|428.91||1.243 ± 0.012|
|561.15||1.555 ± 0.003|
Table 1: DNA Concentrations and Resultant Absorbance Values
Since absorbance with respect to concentration follows the Beer-Lambert Law,
expected absorbance values were calculated from the DNA solution concentrations. Literature values of ε for dsDNA are listed as 0.020µg/ml*cm.
Experimental data can be found in Figure 1 with the calculated absorbance measurements indicated by a solid line. Absorbance measurements are expressed at a 260nm wavelength. Deviation from the theoretical value at higher absorbance values is a result of stray lightinterference within the spectrometer.
Figure 1: Measured versus theoretical abosrbance.
Figure 2: Typical DNA measurement (281.6µg/mL).