Micro Spectrophotometer common sense

When we obtain the results of the nano600Micro Spectrophotometer, it is very important for the correct analysis of the spectrum.
Meaning of the output:
1.A260nm -- the absorption wavelength of the highest absorption peak of nucleic acid.
2.A280nm -- the absorption wavelength of the highest absorption peak of protein.
3.A230nm -- is the absorption wavelength of the highest absorption peak of carbohydrates.
4.A340nm -- is the baseline calibration wavelength for the detection of turbidity and other interference factors of the solution sample. The value should be close to 0.0. If not, it indicates the presence of suspended solids in the solution and the sample needs to be purified. A340 for a pure sample is usually 0.
● The negative value of A230 is mainly due to the interference of some other components in a solution with very low DNA concentration. In the next assay, the dilution of the sample is reduced and the negative value of A230 is corrected.
● The A260/A280 ratio can be used to evaluate the purity of nucleic acid samples: the A260/A280 ratio of pure DNA is 1.8, and the A260/A280 ratio of pure RNA is 2.0. A low ratio indicates contamination with proteins (aromatic) or phenols and requires purification of the sample.
● The A260/A230 ratio can be used to evaluate the purity of nucleic acid samples: the A260/A230 ratio for pure DNA and RNA is 1.8 -- 2.2. A ratio of less than 1.8 indicates contamination with carbohydrates (sugars), salts or organic solvents and requires purification of the sample. When 260/230 & lt; At 1, there are usually only two cases. One is guanidine contamination, the other is protein contamination.
Influence of different sample extraction methods on detection:
● phenol/Trizol extraction -- residual reagent contamination can be indicated by anomalous spectra between 220 and 240nm and offset in the 260 to 280nm region. Do not take too much supernatant after adding chloroform centrifugation, as it is easy to be contaminated by protein.
● Purified column extraction -- Residual guanidin may contribute to the peak near 230nm and trough excursion from 230nm to 240nm.
● Magnetic bead extraction -- residual beads may cause light scattering and lead to abnormal spectra.
1.A260/A280 and A260/A230 ratios are affected by the pH and ionic strength of the solution, such as acid solution will reduce the RATIO of A260/A280, low ionic strength and low pH solution will increase the light absorption value at 280 nm. Therefore, it is necessary to ensure that the ion concentration and pH value of the Buffer used for blank detection and dissolution of samples are consistent.
2. Samples at concentrations close to 2 ng/μ L may result in unreliable 260/280 and/or 260/230 ratios.
3. The dilution concentration of the sample is also a factor that can not be ignored, because there are inevitably some fine particles in the sample, especially the nucleic acid sample. The presence of these small particles can interfere with the effectiveness of the test. The concentration of the sample should not be too low or too high (beyond the test range of the photometer).
4. Sample mixing should be sufficient, otherwise the absorption value is too low, or even negative; There should be no bubbles in the mixture and no suspended matter in the blank liquid, otherwise the reading will drift violently. Uneven sample mixing will directly lead to uncertainty and low repeatability.
5. The sample loading amount should not be too small, and it must be able to connect the upper and lower optical fibers to form an optical column, so that the test can be carried out normally.
6. The sample table shall be cleaned. Before and after the test and after continuo