The electrophoresis detection time of PCR products is generally within 48 h, and some of them are best detected by electrophoresis on the same day. After more than 48 h, the band irregularities disappear. Common problems are as follows: 1. False negative, there is no amplification band. The key steps of PCR reaction are 1 template nucleic acid preparation, 2 primer quality and specificity, 3 enzyme quality and 4 PCR cycle conditions. The reason for finding should also be analyzed and researched in the above links. Template: (1) The template contains heteroproteins (2) The template contains Taq enzyme inhibitors. (3) The proteins in the template are not digested, especially the histones in the chromosomes (4) are too much lost when extracting the template. Or inhalation of phenol (5) template nucleic acid degeneration is not complete. When the quality of the enzyme and the primer is good, there is no amplification band, and it is very likely that the sample is digested, and the template nucleic acid extraction process is out of order. Therefore, an effective and stable digestion treatment solution should be prepared, and the procedure should be fixed and should not be changed at will. . Enzyme inactivation: new enzymes need to be replaced, or both old and new enzymes should be used simultaneously to analyze whether false negatives are caused by loss or insufficient enzyme activity. It should be noted that sometimes you forget to add Taq enzyme or ethidium bromide. Primers: The quality of the primers, the concentration of the primers, and the concentration of the two primers are symmetrical, which is a common cause of PCR failure or unsatisfactory expansion of the bands. Some batches have problems with the quality of primer synthesis. Two primers have a high concentration and a low concentration, resulting in low-efficiency asymmetric amplification. The countermeasures are: (1) Select a good primer synthesis unit. (2) The concentration of the primer should not only look at the OD value, but also pay attention to the primer solution for agarose gel electrophoresis. There must be a primer strip, and the brightness of the two primer strips should be roughly the same, such as a strip with a primer. There is no band in the primer, and PCR may fail at this time, and should be resolved in consultation with the primer synthesis unit. For example, if one primer has high brightness and one has low brightness, the concentration should be balanced when diluting the primer. (3) Primers should be stored in high concentration and small amount to prevent multiple freeze-thaw cycles or long-term storage of refrigerators, resulting in failure of primer degradation. (4) Primer design is unreasonable, such as insufficient primer length, dimer formation between primers, and the like. Mg2+ concentration: Mg2+ ion concentration has a great influence on PCR amplification efficiency. Too high concentration can reduce the specificity of PCR amplification. If the concentration is too low, it will affect the PCR amplification yield and even the PCR amplification will not be amplified. Change in reaction volume: The volume used for PCR amplification is usually 20 ul, 30 ul, 50 ul. Or 100 ul, the application of large volume for PCR amplification, is set according to the different purposes of scientific research and clinical testing. After making a small volume, such as 20 ul, and then making a large volume, it is necessary to mold the condition, otherwise it is easy to fail. Physical reasons: Denaturation is very important for PCR amplification, such as low denaturation temperature, short denaturation time, and high probability of false negatives. Annealing temperature is too low, which can cause non-specific amplification and reduce specific amplification efficiency. High-intensity primers bind to the template to reduce PCR amplification efficiency. It is sometimes necessary to use a standard thermometer to detect denaturation, annealing and extension temperatures in the instrument or water bath, which is one of the reasons for PCR failure. Target sequence variation: If the target sequence is mutated or deleted, it affects the specific binding of the primer to the template, or the primer and the template lose complementary sequence due to a certain deletion of the target sequence, and the PCR amplification is not successful. Second, the PCR amplification bands appearing in the false positive are consistent with the target target sequence bands, and sometimes the bands are more tidy and the brightness is higher. The primer design is not suitable: the selected amplified sequence has homology with the non-target amplified sequence, and thus, when PCR amplification is performed, the amplified PCR product is a non-target sequence. The target sequence is too short or the primer is too short to be prone to false positives. Primers need to be redesigned. Cross-contamination of target sequences or amplification products: There are two reasons for this contamination: (1) cross-contamination of the entire genome or large fragments, resulting in false positives. This false positive can be solved by the following methods: Care should be taken to prevent the target sequence from being drawn into the sample gun or spilled out of the centrifuge tube. All reagents or equipment should be autoclaved except for enzymes and substances that cannot withstand high temperatures. The centrifuge tube and sample tip used should be used at one time. If necessary, the reaction tubes and reagents are irradiated with ultraviolet light to destroy the nucleic acid present before the specimen is added. (2) It is a small fragment of nucleic acid contamination in the air. These small fragments are shorter than the target sequence, but have some homology. Can be spliced ​​to each other, and complementary to the primers, the PCR product can be amplified, resulting in the generation of false positives, which can be alleviated or eliminated by nested PCR. 3. The band appearing after non-specific amplification band PCR amplification is inconsistent with the expected size, large or small, or both specific amplification bands and non-specific amplification bands. The emergence of non-specific bands. Reasons: (1) The primer is not completely complementary to the target sequence, or the primer polymerizes to form a dimer. (2) The Mg2+ ion concentration is too high, the annealing temperature is too low, and the number of PCR cycles is too high. The second is the quality and quantity of the enzyme. Sometimes the enzymes of some sources are prone to non-specific bands and the enzymes of another source do not. Excessive amounts of enzymes sometimes lead to non-specific amplification. Countermeasure: Redesign primers if necessary. Reduce the amount of enzyme or exchange enzymes from another source. Reduce the amount of primers, increase the amount of template, and reduce the number of cycles. Appropriately increase the annealing temperature or use two temperature point method (denaturation at 93 °C, annealing and extension at around 65 °C). Fourth, the appearance of a sheet tow or smear with PCR amplification sometimes occurs with a smear or strip or carpet strip. Cause: The amount of enzyme is too large or the quality of the enzyme is poor, the concentration of dNTP is too high, the concentration of Mg2+ is too high, the annealing temperature is too low, and the number of cycles is too high. Countermeasures: 1. Reduce the amount of enzyme, or exchange enzyme 2 from another source to reduce the concentration of dNTP. Properly reduce the Mg2+ concentration. Increase the amount of templates and reduce the number of cycles.
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