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Nucleic Acid Applications
Nucleic Acid Quantification
It is well established that for determination nucleic acid concentration in solution the absorbance at wavelength 260 nm (A260) is used. The function describing the concentration to absorbance relation is the Lambert-Beer Law: A = e * c * d.
The absorbance (A) is the product of the substance specific extinction coefficient (e), the concentration of the absorbing sample (c), and the optical pathlength in cm (d). A solution of dsDNA in a 10 mm pathlength cell with an optical density of 1.0 has a concentration of 50 µg/ml.
The NanoPhotometer™ Pearl uses the factors 50, 40, 37 and 33 as defaults for dsDNA, ssDNA, RNA and oligonucleotides, respectively, and compensates factors for dilution and varying pathlength.
Nucleic Acid Estimation of Purity
Depending on the extraction/purification or synthesis/purification method of the nucleic acids different impurities can be expected (TRIzol, humic acids, carbohydrates, Guanidine thiocyanate, nucleotides, peptides, EDTA, phenol and protein). It is recommended to include OD ratio measurement (A260/A280 and A260/A230) for purity estimation.
Ratio A260/A280
In solution, pure DNA and RNA typically have A260/A280 ratios of 1.8 and 2.0. If the absorbance ratio is significantly less, the nucleic acid is probably contaminated with protein. Accurate quantification of nucleic acid is not reliable without prior purification, and the efficacy of this can be judged by the A260 /A280 ratio.
Ratio A260/A230
For RNA samples the ratio values ‹ 2.0 point out genomic DNA contamination. Successful DNase I treatment displays in ratio values › 2.0.
Ratio values ‹ 1.5 indicate impurities of extraction chemicals or incompletely removed constituents of cells.
Note: Both ratio values can also be perturbed easily by pH, even if the nucleic acid samples are clean. Use buffers around 7.5 for your measurements.
Use of Background Correction
Background correction at a wavelength totally separate from the nucleic acid and protein peaks at 260 and 280nm, 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.
The NanoPhotometer™ Pearl offers the option of background correction at 320 nm for nucleic acid determination.
Dye incorporation rate
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.
FOI = C(dye) / C(nucleic acid)
Example: Frequency of Incorporation (FOI) of Cy3 per 1000 bases:
FOI(Cy3) = 58.5 * A550/A260
The user can switch between two options of result presentation: a data table or a scan plot.
Use of Background Correction
The absorbance reading of dye labelled nucleic acids at 260 nm is affected by the dye contribution. To obtain accurate concentration values, the contribution has to be eliminated using correction factors.
The NanoPhotometer™ Pearl offers the option to select the background correction for dye contribution of absorbance reading at 260 nm for nucleic acid determination.
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