COMMON PROBLEM
What should I do if a false positive is detected by real-time PCR of African swine fever?
The main reasons are: laboratory contamination, sample or bubble interference, and unstable baseline caused by voltage instability. African swine fever virus infection is a very high viral load in blood and tissue, and samples with a CT value of around 15 are often detected. Centrifugation is required in the process of sample processing and extraction, which may easily cause aerosol contamination during centrifugation of high-concentration samples. Solution: strengthen personnel training, develop good experimental habits, establish an effective communication and incentive mechanism, the fluorescence quantitative PCR laboratory is divided into reagent preparation area, sample processing area, nucleic acid amplification area, strict partition management, pipetting in different areas The devices are distinguished by different identifiers to prevent mixed use.
What should I do if the power is suddenly cut off while the PCR machine is running?
Generally, we recommend configuring a USP power supply in the laboratory, which can ensure that the PCR instrument can be powered normally during operation, thereby ensuring the normal operation of the program. If the thermal cycler is suddenly powered off during operation, if the instrument has a power-off protection function, you can directly continue to run the program. If there is no USP power supply, the PCR program cannot be completed due to a sudden power failure. In order to ensure the authenticity of the experimental results, it is recommended to repeat the experiment and then test it on the computer.
What should I do if the magnetic beads are agglomerated?
The reason for the unity:
1. Caused by the characteristics of the magnetic beads themselves
2. It is caused by the reagent environment where the magnetic beads are located.
3. A large amount of nucleic acid is adsorbed on the surface of the magnetic beads.
4. Too many impurities
Solution: Modify the reagent formula and optimize the reagent preparation to remove impurities.
low nucleic acid yield
There is a problem in the lysis link (mainly because the magnetic beads are not united when they are combined)
There is a problem in the elution process (mainly because the agglomerated magnetic beads are not broken up)
1. Replace the eluent and strictly follow the SOP operation process;
2. Intensify the vibration of the magnetic beads or use a suction tip to suck and stir.
low nucleic acid purity
A260/A280 should be between 1.7-2.0. If it is lower than 1.7, there will be protein contamination. If it is higher than 2.0, there will be RNA contamination. In this case, the cleavage link needs to be optimized. If A260/A230 is less than 1.5, it is considered that there is pollution due to salt pollution, and the washing process needs to be optimized. There may also be alcohol contamination, and the drying time needs to be optimized.
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