Molecular Biology: A Practical Guide - Velikov V.A. 2013

Spectrophotometry of DNA, RNA, and protein preparations
Spectrophotometric determination of DNA concentration

As a first approximation, both the quantity and quality of an isolated DNA preparation are quite often evaluated using gel Electrophoresis, which typically follows immediately after the isolation Procedure. To do this, the ultraviolet fluorescence intensity of the resulting sample in an adjacent gel lane is visually compared with that of a sample of known concentration.

The DNA concentration, as well as its degree of purity, can be determined more accurately and rapidly using a spectrophotometer. This is done by measuring the optical density of the DNA solution at a wavelength of 260 nm. One (each) optical unit corresponds to a DNA concentration of 50 µg/mL. For calculations, you can use the calculator available on the MOLBIOL.RU website (http://www.molbiol.ru) or similar portals.

(Absorption) spectrophotometry is a physicochemical method for investigating solutions and solid substances, based on The Study of absorption spectra in the ultraviolet (200-400 nm), visible (400-760 nm), and infrared (>760 nm) spectral regions. The fundamental relationship studied in spectrophotometry is the dependence of the absorption intensity of incident light on the wavelength λ. According to the Beer-Lambert law, the optical density of a solution is directly proportional to the concentration of the absorbing substance. Nucleic Acids (NAs) absorb UV radiation in the 240-290 nm range, with a maximum at 260 nm. The chromophores are the nitrogenous bases of NAs, particularly Pyrimidines. Pyrimidines absorb UV light approximately 10-20 times more intensely than the chromophores of protein molecules—Tryptophan, Tyrosine, and phenylalanine.

To assess the purity of an RNA-free DNA preparation, optical density measurements of the solution are carried out at wavelengths of 260, 280, and 235 nm—corresponding to the absorption maxima of DNA, protein, and polysaccharide solutions, respectively. The A_260/280 ratio for pure DNA should be greater than 1.8, and the A_260/235 value should exceed 2.2. Polysaccharide contamination is characteristic mainly of plant DNA preparations. Unlike other CARBOHYDRATES, Lignin contains aromatic atomic groups and therefore absorbs UV radiation.

The lower concentration limit of DNA that can be determined spectrophotometrically is 0.1 µg/mL. Typically, an aliquot (Lat. aliquoties — several parts, multiple) of the test DNA solution, for example, 1 µL, is taken for the assay and diluted 100-fold or more.

The resulting solution concentration is then recalculated. It is important that the diluted sample contains more than 10 ng of DNA. For comparison, gel electrophoresis can also visualize a band containing as little as 10 ng of DNA. The DNA sample must be free of RNA.

Materials and Equipment

Genomic DNA from Bacteria, Blood Cells, or corn Tissues of unknown concentration, a spectrophotometer, and a DNA sample of any origin with a known concentration.

Solutions

- DNA solution of unknown concentration in TE buffer.

- Lambda phage DNA solution in TE buffer at a concentration of 1 mg/mL.

Procedure

1. Using a micropipette, take 1 µL of the obtained DNA sample for analysis and dilute the preparation by adding 130 µL of TE buffer containing 100 mM NaCl ^a. The sample volume is adjusted to 130 µL so that the curvature of the liquid surface does not affect the measurements.

2. Place the diluted DNA sample into the "small" (100 µL) cuvette.

3. Measure the A₂₆₀ absorbance. The resulting value should lie within the range of 0.005-2.5 ^b. Otherwise, the DNA must be diluted or concentrated ^c.

4. Calculate the DNA concentration using the conversion factor from Table 1 according to the formula: C [µg/mL] = A₂₆₀ × K

Class="center">Table 1. Calculation of DNA and RNA concentration

5. Measure the A₂₈₀ and A₂₃₅ absorbances to evaluate the efficiency of DNA purification from protein and polysaccharide contaminants. Both the 260/280 and 260/235 ratios should be greater than 1.8. Pure DNA is characterized by A₂₆₀/A₂₈₀ = 1.8–1.9 and A₂₆₀/A₂₃₅ = 2.2–2.5. For pure RNA, the A₂₆₀/A₂₈₀ value is 1.9–2.0 ^d

6. Open the homepage of the MOLBIOL.RU website (http://www.molbiol.ru). On the main page, find the "Calculations" section, select "Spectrophotometric determination of DNA (RNA) concentration," and calculate the concentration of your sample using the dedicated form.

Notes

^a Other low-salt buffers can be used to dissolve DNA (RNA), but Water must be avoided. For instance, solutions of 100 mM NaCl, 20 mM Na₃PO₄, 10-100 mM Tris-HCl (pH 7.5-9.0), or 100 mM K₂HPO₄ (pH 8.2) yield similar results. Measurements in water lead to significant deviations. The measurement error can reach up to 14%, and the A₂₆₀/A₂₈₀ ratio turns out to be underestimated.

^b Measurement accuracy decreases at excessively high and low A₂₆₀ values due to deviations from the Light absorption law. The error at a value of 0.05 is ≈ 18%, and at 0.1-1.0 it is ≈ 1%. Measurements taken at values exceeding 2.5 are unreliable.

^c DNA is concentrated by ethanol reprecipitation (Protocol 1.4).

^d For oligonucleotides, whose absorption noticeably depends on their base composition, special calculations exist.

^d These values are valid if the measurement is carried out in a buffer solution (e.g., TE) at a neutral pH.