Nucleic Acids

Nucleic Acid quantification is an important step in many different life science protocols including those implemented for NGS, qPCR, and many other areas. The Nucleic Acid Spectrophotometer, NanoPhotometer® by Implen, offers a preprogrammed Nucleic Acid method with options to measure all types of nucleic acids including dsDNA, ssDNA, RNA, miRNA, Oligo, options to enter sequences for miRNAs and Oligos as well as a custom-defined extinction coefficient for other types of nucleic acids. It is also possible to measure labeled nucleic acids via the absorbance maxima of the dye. Dye concentration and the frequency of incorporation are calculated and displayed automatically.

Measuring Nucleic Acid Concentrations

With the NanoPhotometer®, one of the top Nucleic Acid Spectrophotometers, the concentrations and purity of a nucleic acid sample is easily determined over a vast dynamic range without tedious and error prone manual dilution steps before the reading. There are also no pricy consumables needed, and cleaning is simply done with a wipe of the measurement head quartz surfaces. No reconditioning of these surfaces, no recalibration and no maintenance of the instrument is required ever.

The UV/Vis spectrophotometric analysis of nucleic acid samples is based on a reading at 260 nm using the Beer-Lambert law. An unknown concentration can easily be calculated by multiplying the optical density of the sample at 260 nm with the extinction coefficient of the nucleic acid present in the solution. As a general rule, OD 1 measured on a spectrophotometer can be translated to:

dsDNA = 50 ng/µl
ssDNA = 37 ng/µl
RNA = 40 ng/µl
where the optical density is defined as

OD = Log (Intensity of the incoming light / Intensity of outgoing light)

Wavelengths for Measuring Nucleic Acid Concentrations

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The Beer-Lambert law, also known as Beer’s Law, empirically relates the absorption of light to the properties of the sample. This law states that there is a logarithmic relationship between the transmission of light through a specific sample (T = I/Io with I = outgoing light and Io = incoming light), the molar extinction coefficient for a specific compound (ε), the concentration of the absorbing species in the material (c) and the distance the light travels (d).

Impurities of a nucleic acid sample can be assessed by adding several wavelengths to each reading (230, 280 and 320 nm). The ratio of 260/280 will give an indication of how much protein is present in the sample. For dsDNA, the 260/280 ratio should be around 1.8, for RNA samples a ratio of around 2.0 is indicating that protein contaminations are minimal.

The 260/230 ratio should be around 1.8 to 2.2 for purified samples. Low 260/230 ratios can be expected for residual phenol or guanidine in the solution, high ratios are indicating an inappropriate solution for the blank measurement.

A reading higher than 5 mAbs at 320 nm is indicating that either salt residues or undissolved particles are present in the sample.

implen-nanophotometer-NP80-for-nucleic-acid-measurement-applications-nanodrop-alternative-and-warning-screen

Nucleic Acid Quantification with NanoPhotometer® NP80

Let’s cover the performance of the NanoPhotometer® NP80, top Nucleic Acid Spectrophotometer on the market, 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. The Beer-Lambert law can be applied to determine the nucleic acid concentration in a sample. Along with the nucleic acid concentration, the Nucleic Acid spectrophotometer NanoPhotometer® NP80 also assesses the purity of the nucleic acid sample by calculating the 260/230 and 260/280 ratios. The NanoPhotometer® features Blank Control™ to automatically warn the user if high background is present in a blank from buffers or contaminants.

Material & Methods

DNA from fish sperm 74782 from Sigma (Lot. 1343783 was used to create aqueous stock solution with 30,000 ng/µL. Different sample concentrations were achieved by dilution with distilled water. Dilution ratio was controlled by weight via microbalance (Sartorius BP221S). Expected absorbance values were determined in 10 mm quartz glass cuvettes (Hellma Analytics 100-QS) with a calibrated UV/Vis spectrometer UVIKON XL (serial number 110178). All sample measurements were done using the Nucleic Acid Spectrophotometer, NanoPhotometer® NP80 (serial number M80798).

DNA concentrations were measured five times using a sample volume of 1 µl. Samples were vortexed before each measurement to ensure homogeneity. After each measurement, the pedestal and the lid of the Nucleic Acid Spectrophotometer were cleaned with a slightly wet lint free tissue and a new aliquot of the sample was pipetted.

implen-nanophotometer-NP80-for-protein assay-measurement-applications-nanodrop-alternative-cleaning-lid

Precision and Accuracy of the Nucleic Acid Spectrophotometer Measurement Results

Accuracy Results

Table 1 demonstrates the expected DNA concentration and the mean value of the five measurements of each DNA concentration. Additionally, the standard deviation of the mean absorbance at the 0.67 mm / 0.07 mm path of each sample is listed.

Ratio

The Nucleic Acid Spectrophotometer, NanoPhotometer® NP80, calculates the 260/230 and 260/280 ratios which give information about contaminants of the sample. The 260/230 ratio should be > 1.8, lower ratios indicate contamination with e.g. guanidinium thiocyanate or other buffer salts (TRIS, EDTA) used during the nucleic acid isolation/purification. The 260/280 ratio indicates the presence of proteins in the nucleic acid sample. Pure DNA and RNA preparations have expected ratios of ≥ 1.8 and ≥ 2.0, respectively.

Linearity Results

The resulting linearity curve in the range of 1.89 – 14,711 ng/µl shows a close correlation between expected and measured concentrations with a coefficient of determination (R2) of 0.9998 (Figure 1).

Figure 1:
Samples (mean value) in the range of
1.89 – 14,711 ng/µl were measured with automatic path length selection.

implen-nanophotometer-nucleic-acid-measurement-applications-nanodrop-alternative-expected-concentration2

Figure 2:
Samples (mean value) in the range of
1.89 – 932 ng/µl
were measured with path length 0.67 mm.

implen-nanophotometer-nucleic-acid-measurement-applications-nanodrop-alternative-expected-concentration3

Figure 3:
Samples (mean value) in the range of
1,863.9 – 14,711 ng/µl were measured with path length 0.07 mm.

Accuracy and Linearity of the NanoPhotometer®, Nucleic Acid Spectrophotometer

The presented data demonstrate the high accuracy and linearity of the NanoPhotometer® across the entire dynamic range of the instrument. With this novel approach of only two precise path lengths with fixed anchor points utilizing the proprietary True Path Technology™ in a sealed optical environment, mechanical changes in the system are eliminated.

For further technical information please refer also to Technical Note #1 True Path Technology™.

The NanoPhotometer® is the only Nucleic Acid Spectrophotometer with True Path Technology™ providing accurate results without the need for recalibration throughout the entire lifetime of the instrument.

NanoPhotometer® Models that Work Best for Nucleic Acid Quantification

All units are battery powered and require NO maintenance or recalibration for a lifetime. They also can be used for the following applications: dsDNA, ssDNA, RNA, mRNA, miRNA, Oligos, Proteins and Antibodies including dye labeled samples, small molecules in organic solvents, kinetics in a drop and more.

implen 12-channel nanophotometer N120 for nucleic acid measurements, nanodrop alternative

NanoPhotometer®
N120

Microvolume High Throughput Spectroscopy

• High Performance Microvolume Spectrophotometer • Only 1.7 sec per sample, 20 sec per run (12 samples) • Full high resolution scan from 200-900 nm for each sample
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implen cuvette nanophotometer NP80 for nucleic acid measurements, nanodrop alternative

NanoPhotometer®
NP80

All-in-One
Spectroscopy

• All-in-One Spectrophotometer
• Micro-volume and cuvette capability • Built-in vortexer
• Full high resolution scan from 200-900 nm for each sample     • Temperature Controlled Cuvette Holder • Starting at 0.3 µl sample volume
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implen cuvette nanophotometer C40 for nucleic acid measurements, nanodrop alternative

NanoPhotometer®
N60*/N50

Microvolume
Spectroscopy

• High Performance Micro Volume Spectrophotometer
• Starting at 0.3 µl sample volume • Full high resolution scan from 200-900 nm for each sample* • Built-in vortexer*
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implen nanophotometer C40 for nucleic acid measurements

NanoPhotometer®
C40

Cuvette
Spectroscopy

• Standard and Microvolume cuvettes (quartz, glass and plastic) • Additional Applications: OD600 (Cell, Bacterial, Yeast Density),
Bradford, BCA • Temperature Controlled Cuvette Holder
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“Best small volume spec on the market”

Rating: 5.0  ★★★★★

Application Area: Protein/nucleic acid quantitation

“The Implen NanoPhotometer N50 is extremely easy to use (intuitive menus and settings), accurate, and genuinely capable of measuring very small volumes repeatably. The touch screen works well even with gloves. Window configurations are adaptable so you can customize the information you’re looking at while working. The instrument is light and mobile, and since it’s all-in-one, you can literally move it to where you’re working as needed. It’s not stuck in a particular corner of the lab. In my opinion, it’s the best nano-scale measurement device on the market for routine lab sample quantification and spectral reading.”

David Rawling Organization: Inflammatix, Inc., USA

“Easy use and accurate results “

Rating: 4.7  ★★★★★

“We are using it for nucleic acid quantification. It’s so easy to use, gives accurate results and easy to save data in an organized way. Would recommend it.”

Dr. Assala Samad Organization: University of Oxford, United Kingdom

“Small but excellent”

Rating: 5.0  ★★★★★

Application Area: Gastroenterologie/Hepatologie, Medizinisches Forschungszentrum

“The N60 is an excellent product for determining concentrations of nucleic acids and proteins. Advantages include the accessibility of measured data, as the device exports measurements as .csv and .pdf files and that the device can be cleaned with 70% Ethanol and water. Plus, it contains an in-built vortexer.”

Dr. Sabrina Rüschenbaum Organization: Universitätsklinikum Essen AöR, Germany

“Great accuracy of measurements, easy usage, really nice interface.”

Rating: 5.0   ★★★★★

Application Area: determination of the concentration of proteins and nucleic acids solutions

“C40 NanoPhotometer is a great tool to measure macromolecule concentrations in microvolumes. It surpasses our previous nanodrop machine as it does not need calibration and has more functions. Easy interface is a great advantage and the new function of enzyme kinetics together with vortex makes this small and handy tool a multitask help in our lab. The wi-fi functions enables us to print spectra quickly. Measurements are repeatable and accurate. I would recommend the equipment to every biochemistry lab.”

Joanna Sliwiak Organization: Institute of Bioorganic Chemistry, Laboratory of Protein Engineering, USA

“Producto NP80-Touch”

Rating: 5.0  ★★★★★

Application Area: Biologia celular y desarrollo

Es un buen producto que facilita con rapidez y eficacia la medición de ácidos nucleicos y proteínas.”

Dr. Jesus Aguirre Organization: Instituto de Fisiologia Celular, Mexico