Visible spectrophotometer can be used for nucleic acid quantification

Spectrophotometer, visible spectrophotometer is used for nucleic acid quantification. The spectrophotometer uses a light source that can generate multiple wavelengths. Through a series of spectrometers, a light source of a specific wavelength is generated. After the light source passes through the tested sample, part of the light source is Absorb, calculate the absorbance value of the sample, which is converted into the concentration of the sample. The absorbance of the sample is proportional to the concentration of the sample.

Quantification of nucleic acids

The quantification of nucleic acid is the most frequently used function of spectrophotometer. It can quantify oligonucleotides, single-stranded and double-stranded DNA, and RNA dissolved in buffer. The absorption wavelength of the highest absorption peak of nucleic acid is 260 nm. Each nucleic acid has a different molecular composition, so its conversion factor is different. To quantify different types of nucleic acids, the corresponding coefficients must be selected in advance. For example: 1OD absorbance value is equivalent to 50μg / ml dsDNA, 37μg / ml ssDNA, 40μg / ml RNA, 30μg / ml Olig. The absorbance value after the test is converted by the above-mentioned coefficients to obtain the corresponding sample concentration. Before testing, select the correct program, enter the volume of the original solution and diluent, and then test the blank solution and sample solution. However, the experiment was not smooth. Unstable readings may be the biggest headache for experimenters. The higher the sensitivity of the instrument, the greater the shift in absorbance. In fact, the design principle and working principle of the spectrophotometer allow the absorbance value to change within a certain range, that is, the instrument has a certain accuracy and precision. For example, the accuracy of Eppendorf Biophotometer is ≤1.0% (1A). It is normal for the results of such multiple tests to vary between about 1.0% average. In addition, the physical and chemical properties of the nucleic acid itself and the pH value and ion concentration of the nucleic acid-dissolving buffer should also be considered: during the test, too high ion concentration will also cause the reading to drift, so it is recommended to use a certain pH value and low ion concentration Buffers, such as TE, can greatly stabilize readings. The dilution concentration of the sample is also a factor that cannot be ignored: due to the inevitable presence of some fine particles in the sample, especially nucleic acid samples. The presence of these small particles interferes with the test effect. In order to minimize the impact of the particles on the test results, it is required that the nucleic acid absorbance value is at least greater than 0.1A, and the absorbance value is preferably 0.1-1.5A. Within this range, the interference of the particles is relatively small, and the results are stable. This means that the concentration of the sample cannot be too low or too high (beyond the test range of the photometer). Finally, there are operational factors, such as sufficient mixing, otherwise the absorbance value is too low, or even a negative value; there can be no bubbles in the mixed liquid, no suspension in the blank liquid, otherwise the reading drifts drastically; the blank and sample must be tested with the same cuvette Otherwise, the concentration difference is too large; the conversion coefficient and the sample concentration unit are selected to be consistent; the cuvette with window wear cannot be used; the volume of the sample must reach the minimum volume required by the cuvette and other operational matters.

In addition to the nucleic acid concentration, the spectrophotometer also displays several very important ratios indicating the purity of the sample, such as the A260 / A280 ratio, which is used to evaluate the purity of the sample because the absorption peak of the protein is 280 nm. For pure samples, the ratio is greater than 1.8 (DNA) or 2.0 (RNA). If the ratio is lower than 1.8 or 2.0, it indicates the presence of protein or phenols. A230 means that there are some contaminants in the sample, such as carbohydrates, peptides, phenol, etc., the ratio of the pure nucleic acid A260 / A230 is greater than 2.0. A320 detects the turbidity of the solution and other interference factors. Pure samples, A320 is generally 0.