Nucleic Acid quantification is an important step in many different life science protocols including those implemented for sequencing and other areas. In this application note, we describe the performance of the NanoPhotometer® NP80 in terms of linearity and accuracy, applying small-volume nucleic acid quantification at 260 nm. Nucleic acid samples display a characteristic absorption spectrum at 260 nm. Therefore, the Lambert-Beer law can be applied to determine the nucleic acid concentration in a sample. Along with the nucleic acid concentration, the NanoPhotometer® NP80 also assesses the purity of the nucleic acid sample by calculating the 260:230 and 260:280 ratios.
The NP80 Spectrophotometer provides the smallest sample volume option with as little as 0.3 µl of sample
Technical specifications of the NanoPhotometer® NP80 Spectrophotometer
The NanoPhotometer® NP80 is a full scanning spectrophotometer (200-900 nm) with the option for small volume option that allows for reliable measurements in a minimum sample volume starting at 0.3 μl. The sample is squeezed to exactly defined path lengths with a patented Sample Compression Technology™ which is independent from surface tension. In addition, this technology avoids evaporation, allows the sample to be easily retrieved, and allows for reliable quantification of proteins dissolved in volatile solvents like toluene. True Path Technology™ utilizes two fixed anchor points for a wide concentration range from 1 ng/µl to 16,500 ng/µl of dsDNA and guarantees exact pathlength over the lifetime of the instrument. Therefore, the NanoPhotometer® NP80 is recalibration and maintenance free.
Nucleic Acid Quantification Linearity and Precision
As shown previously with NanoPhotometer® instruments, the NanoPhotometer® NP80 also features excellent accuracy regarding low-volume nucleic acid quantification. The linearity and the reproducibility of different nucleic acid quantification methods were verified with the NanoPhotometer® NP80 utilizing oligonucleotide samples in concentrations ranging from 2 ng/µl to 2,000 ng/µl. The measurements were performed in triplicate for each oligonucleotide sample without prior dilution with a sample volume of 1.0 μl. The samples were vortexed before each measurement to ensure sample homogeneity. After each measurement, the measurement window and the mirror in the lid were cleaned with a slightly wet, fluff-free tissue, and a new aliquot of the sample was pipetted.
The NanoPhotometer® NP80 produced linear and highly reproducible measurements with minimal standard deviation between triplicates
Results
We have shown that the NanoPhotometer® NP80 is highly reliable for nucleic acid quantification. The resulting linearity curve shows a close correlation between expected and measured concentrations with coefficient of determination (R2), of 0.9997. According to the reproducibility results for the nucleic acid quantification method, the standard deviation for the repeated measurements is minimal and emphasizes the high accuracy of the whole application. The microvolume option of the NanoPhotometer® NP80 is therefore suited for laboratories where sample volumes are limited, a fast answer without prior dilution is needed, and where retrieval of the sample for subsequent applications is required.
Samples were vortexed prior to each measurement with a low-vibration vortexer. The sample window and lid were cleaned after each measurement with a fluff-free tissue.