1. No PCR amplification product bands after agarose gel electrophoresis

Main manifestations: Marker and control bands are normal, but the test sample has no bands.

Reason analysis: 1. Inappropriate reaction conditions: the annealing temperature is too high during the PCR process, the annealing extension time is insufficient, and the number of reaction cycles is too small.

                 2. Low concentration or purity of nucleic acid template: The detection sensitivity of ordinary PCR is limited, and the sample nucleic acid content is low or contains inhibitors.

                 3. The reaction system does not match the nucleic acid amplification: the Mg2+ concentration in the detection system, the amount of dNTPs or the Buffer does not match the conditions required for the target nucleic acid amplification.

                 4. Improperly designed or degraded primers: primer-dimers or secondary structures exist, unsuitable storage conditions after primer dilution, or excessive freeze-thaw cycles.

Solution: 1. Optimize the reaction conditions, explore the annealing temperature gradiently, prolong the annealing extension time, and increase the number of reaction cycles (30-45 range is appropriate).

                 2. Purify the nucleic acid template, or use a commercial nucleic acid extraction kit for extraction to increase the amount of nucleic acid template loaded.

                 3. Optimize the reaction system (except the premixed system), change the Mg2+ concentration and the amount of dNTPs in the detection system, and replace the Buffer in the system.

                 4. Redesign primers to avoid primer-dimers and intra-chain secondary structures. After one dilution of primers, it can be divided into multiple small tubes to reduce the number of repeated freezing and thawing.