Common uses of Micro Spectrophotometer
Micro Spectrophotometer has become a routine instrument in modern molecular biology laboratory. It is often used for the determination of nucleic acid, protein and bacterial growth concentration.
Quantification of nucleic acid
Nucleic acid quantification is a high frequency function of the Micro Spectrophotometer. Oligonucleotides, single - and double-stranded DNA, and RNA soluble in buffers can be quantified. The absorption wavelength of the high absorption peak of nucleic acid is 260nm. Each nucleic acid has a different molecular composition, so its conversion coefficient is different. To quantify different types of nucleic acids, the corresponding coefficients should be selected in advance. For example, the absorption value of 1OD is equivalent to 50μg/ mL of dsDNA, 37μg/ mL of ssDNA, 40μg/ mL of RNA, 30μg/ mL of OLIG. The absorption value after the test is converted by the above coefficients to obtain the corresponding sample concentration. Before the test, select the correct procedure, input the volume of the original solution and diluent, and then test the blank solution and sample solution. However, the experiment was not smooth sailing. Unsteady readings can be a headache for the experimenter. The higher the sensitivity of the instrument, the greater the shift of absorption value shown.
For example, the accuracy of the Colibrimicro Spectrophotometer in Berger, Germany is less than 1.0% (1A). It is normal for the results of these tests to vary by an average of around 1.0%. In addition, it is also necessary to consider the physical and chemical properties of the nucleic acid itself and the pH value of the buffer solution to dissolve the nucleic acid, ion concentration and so on: in the test, the ion concentration is too high, will also lead to reading drift, so it is recommended to use a certain pH value, low ion concentration of buffer solution, such as TE, can greatly stabilize the reading. The dilution concentration of the sample is also a factor that cannot be ignored: due to the inevitable existence of some fine particles in the sample, especially the nucleic acid sample. The presence of these small particles interferes with the test effect. In order to reduce the impact of particles on the test results, the light absorption value of nucleic acid is required to be at least greater than 0.1A, and the light absorption value should be 0.1-1. In this range, the interference of particles is relatively small and the results are stable. This means that the concentration of the sample must not be too low, or too high (beyond the range of the photometer). The latter is the operational factors, such as mixing to be sufficient, otherwise the light absorption value is too low, or even negative value; Mixed liquid can not exist bubbles, blank liquid without suspended matter, otherwise the reading drift; The same colorimetric cup must be used to test the blank solution and the sample, otherwise the concentration difference is too great; Conversion coefficient is consistent with sample concentration unit selection. Can not use window wear of the colorimetric cup; The volume of the sample must meet the small volume required by the colorimetric cup and other operational matters.
For pure samples, the ratio is greater than 1.8 (DNA) or 2.0 (RNA). A ratio below 1.8 or 2.0 indicates a protein or phenolic influence. A230 indicates that there are some pollutants in the sample, such as carbohydrates, polypeptides, phenols, etc. The A260/A230 ratio of the relatively pure nucleic acid is greater than 2.0. A320 detects the turbidity and other interference factors of the solution. For pure samples, A320 is usually 0.
This method directly tests the protein at 280nm. By choosing the Warburg formula, the photometer can display the concentration of the sample directly, or choose the corresponding conversion method to convert the absorption value to the concentration of the sample. The protein determination process is very simple, first test the blank fluid, then test the protein directly. Due to the presence of some impurities in the buffer, it is generally necessary to eliminate the 320nm "background" information and set this function to "on". Similar to the nucleic acid test, the absorbance value of A280 is required to be at least greater than 0.1A, with A linear range of 1.0-1.5. In the experiment, when Warburg formula was selected to display the sample concentration, the reading was found to be "drifting". This is a normal phenomenon. In fact, as long as the A280's absorbance value is observed to vary within a range of 1%, the results are very stable. The reason for the drift is that the absorption value of Warburg formula is converted into concentration and multiplied by a certain coefficient. As long as the absorption value is slightly changed, the concentration will be magnified, which makes the result very unstable. The direct protein quantification method is suitable for the determination of relatively pure protein with relatively single composition. Compared with colorimetric method, UV direct quantitative method is fast and easy to operate. But susceptible to interference by parallel substances, such as DNA; In addition, the sensitivity is low and the protein concentration is high.
Colorimetric methods generally have BCA, Bradford, Lowry and other methods.
Lowry method: Based on and improved on the earlier Biuret reaction. Proteins react with Cu2 to produce blue reactants. However, Lowry's method is more sensitive than Biuret's. The disadvantage is that several different reaction reagents need to be added in sequence; The reaction time is longer; Susceptible to non-protein substances; The proteins containing EDTA, TritonX-100, Ammoniasulfate and other substances are not suitable for this method.