In biological experiments, most of the experimental tests are microscopic, based on cell, molecular, and protein levels, and these microscopic samples are sensitive to external environmental influences, such as temperature, pH, enzymatic hydrolysis, damage, etc., in our efforts to protect the survival of the sample. At the same time of the environment, pollution is also a top priority. It is necessary to resolutely avoid the intrusion of “impurities†in the environment, including sample contamination, reagent instrument cross-contamination, and product aerosol pollution. "Easy to check is not easy to see", the pollution comes quietly, and it is impossible to prevent it. The false positive result makes countless biological people crazy and collapse.
Xiaobian is here today to see what you don't know about the PCR experiments contaminated by biological laboratories.
In many biological laboratories, molecular biology detection methods such as PCR have been widely used due to their high sensitivity, rapidity, and convenient operation. However, due to its high sensitivity, very small amounts of pollution sources may lead to detection results. False positive, so the control of environmental pollution is also extremely strict. In the face of numerous sources of pollution, such as sample cross-contamination, reagent instrument cross-contamination, clonal plasmid contamination, PCR amplification product contamination, and aerosol contamination that is highly negligible, aerosols are dispersed by solid or liquid small particles and suspended in gas. The colloidal dispersion system formed in the medium, when the air and the liquid surface are rubbed, for example, the reaction tube is shaken more vigorously during operation, and the aerosol can be formed when the cover is opened, the sample is sucked, and the repeated injection of the contaminated injection gun can form an aerosol. . It is calculated that an aerosol particle can contain 48,000 copies, so the pollution caused by it is a very important and neglected source of pollution.
Although the impact of these pollutions on the results of PCR experiments has received extensive attention, most laboratories have adopted various methods for pollution control, such as regular cleaning, clearing of operating areas, reagent dispensing, careful operation, repeated experiments, multi-angle verification. result. However, a small amount of nucleic acid fragments can be amplified, so the control of pollutants can not only be eliminated or avoided, but the key is to control from the source. At present, most biological laboratories, molecular experiments are carried out on a large scale every day, pollution sources have been ubiquitous, specific, non-specific, homologous, non-homologous, widely distributed in sample containers, laboratory countertops, reagent solutions In the middle of the instrument, the inner tube of the instrument is attached, etc. Do you think that ordinary cleaning can solve these pollution sources? It must not be done. The labs have developed a variety of methods to eliminate these sources of pollution.
Traditional way | principle | Advantage | insufficient |
Alcohol wipe | Precipitating the nucleic acid and wiping it to ensure that the pollution surface contamination is relieved to some extent. | Simple, convenient, and low cost | Incomplete (the nucleic acid fragment itself does not change, there is still the possibility of aerosol contamination), small pore size equipment can not be removed |
Modification | Marks genetic material, prevents polymerase binding to nucleic acids, thereby inhibiting DNA amplification | Relatively good effect | Reversible reaction, high cost, reagents may have unsafe effects |
Enzymatic hydrolysis | Enzymes degrade long-chain nucleic acid molecules into small fragments | Fast speed and relatively good results | There are still large fragments in the degraded nucleic acid, with complete genetic information, which can be amplified by PCR and the cost is high. |
High pressure denaturation | High pressure can degrade nucleic acids into small fragments of 20-30 bp | Thorough, low cost | Only for high pressure resistant materials, not for experimental stations and large experimental equipment |
Ultraviolet irradiation | The pyrimidine base in the PCR product will form a dimer and terminate the extension. | Convenient, wide range, low cost | It is only effective for long fragments of 500 bp or longer, and has little effect on short fragments, and not all pyrimidines in the DNA strand can form dimers, and UV irradiation can also break the dimers. |
Xiaobian encountered PCR pollution in the laboratory at the beginning, but at the time, I didn’t know that there were such artifacts in the world. The experiment was repeated and tossed many times before I got a successful result. Good things should be shared. I recommend it to everyone, I hope to You are helpful to see the official.
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