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The electrophoresis detection time of the PCR product: generally within 48 hours, some of which are best detected by electrophoresis on the same day, and the band irregularity disappears after more than 48 hours.

False negative, no amplification bands appear.

The key steps of the 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 the problem should also be analyzed and researched on the above links.

Template: 1 template contains heteroprotein, 2 template contains Taq enzyme inhibitor, 3 template protein is not digested, especially histone in chromosome, 4 is excessively lost when extracting template, or inhaled phenol. 5 template nucleic acid denaturation 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 Taq enzyme or ethidium bromide is forgotten.

Primers: Primer quality, concentration of primers, and symmetry of the concentration of the two primers are common causes of PCR failure or unsatisfactory amplification 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 as follows: 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 band, and the brightness of the two primer bands should be roughly the same, such as a primer with a band and a primer without Strips, PCR may fail at this time, and should be resolved in consultation with the primer synthesis unit. If one primer has high brightness and one has low brightness, balance the concentration 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 not reasonable, such as the length of the primer is not enough, the formation of dimers between the primers.

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 100ul, 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 20ul, 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 highly likely 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 template lose the complementary sequence due to the deletion of a certain target sequence, and the PCR amplification is not successful.

False positive: The PCR amplification band that appears is consistent with the target target sequence band, and sometimes the band is more tidy and the brightness is higher.

Primer design is not suitable: the selected amplified sequence has homology with the non-target amplified sequence, so 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: one is cross-contamination of the entire genome or large fragments, leading to false positives. This false positive can be resolved 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. The second is the contamination of small fragments of nucleic acids in the air. These small fragments are shorter than the target sequences, but have some homology. The splicing can be spliced ​​to each other, and after complementing the primers, the PCR product can be amplified, resulting in the generation of false positives, which can be alleviated or eliminated by nested PCR.

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