What are the precautions for PCR experiment operation?

Precautions for experimental operation: Although residual contamination of amplified sequences is mostly the cause of false-positive reactions, cross-contamination between samples is also one of the reasons. Therefore, not only should the amplification reaction be carried out carefully, but also in all steps of sample collection, extraction and amplification:

(1) Wear disposable gloves. If the reaction solution is accidentally splashed, change the gloves immediately.

(2) Use disposable tips. It is strictly forbidden to mix them with the tips in the PCR product analysis room. Do not expose the tips to the air for a long time to avoid aerosol contamination.

(3) To avoid splashing of the reaction liquid, in order to avoid this situation when opening the reaction tube, slightly centrifuge the liquid to collect the liquid at the bottom of the tube before opening the cap. If accidentally spilled on gloves or tabletop, change gloves immediately and wipe the tabletop with dilute acid.

(4) When working with multiple samples, prepare a reaction mixture, first mix dNTPs, buffers, primers and enzymes, and then divide them into packages, which can reduce operations, avoid contamination, and increase the accuracy of the reaction.

(5) Finally, the reaction template was added, and the reaction tube was tightly capped after the addition.

(6) Set up negative and positive controls and blank controls during operation, which can verify the reliability of the PCR reaction and assist in judging the reliability of the amplification system.

(7) Use a replaceable or high-pressure-treated sampler as much as possible. Since the sampler is most likely to be contaminated with by-product aerosols or sample DNA, it is best to use a replaceable or high-pressure-treated sampler. If there is no such special sampler, at least the sampler should be dedicated during the PCR operation and cannot be used interchangeably, especially since the sampler used for PCR product analysis cannot be taken to the other two areas.

(8) Repeat the experiment, verify the results, and draw conclusions with caution