Views: 5 Author: Site Editor Publish Time: 2021-11-25 Origin: Site
When performing PCR operations, the operator should strictly abide by some operating procedures to minimize the possibility of PCR contamination or prevent the occurrence of contamination.
1. Divide the operation area: At present, ordinary PCR can not achieve single-person single-tube and complete closed-tube operation. However, whether it can achieve single-person single-tube or not, the experimental operation is required to be carried out in three different areas. Pre-processing and post-processing should be carried out in different isolation areas:
(1) Specimen processing area, including preparation of amplification templates.
(2) Amplification zone, including preparation of reaction solution and PCR amplification.
(3) Product analysis area, gel electrophoresis analysis, product photography and preparation of recombinant clones.
(4) There must be a certain degree of isolation in each work area, special operation equipment, and a certain directionality. Such as: specimen preparation → PCR amplification → product analysis → product processing.
Remember: Do not take the products and equipment in the product analysis area to the other two working areas.
2. Distributed reagents: The reagents needed for PCR amplification should be prepared and dispensed on the ultra-clean workbench or negative pressure workbench equipped with ultraviolet lamps. All pipettes and pipette tips must be fixed in it, and cannot be used to absorb amplified DNA and DNA from other sources:
(1) The PCR water should be high-pressure double distilled water.
(2) The primers and dNTPs are prepared with high-pressure double-distilled water in the area without PCR amplification products.
(3) The primers and dNTP should be stored separately, and the time should be marked when the aliquots are used to find the cause in case of contamination.
3. Precautions for experimental operation: Although most of the residual contamination of the amplified sequence is the cause of false positive reactions, cross-contamination between samples is also one of the reasons. Therefore, it is not only necessary to be cautious and conscientious in performing amplification reactions, but also to pay attention to all aspects of sample collection, extraction and amplification:
(1) Wear disposable gloves. If the reaction solution is accidentally splashed, replace 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 pollution.
(3) Avoid splashing the reaction liquid. To avoid this situation when opening the reaction tube, centrifuge a little before opening the cap to collect the liquid at the bottom of the tube. If accidentally spilled on the gloves or the table, you should immediately change the gloves and wipe the table with dilute acid.
(4) When working with multiple samples, prepare the reaction mixture by mixing the dNTP, buffer, primer and enzyme first, and then aliquot, which can reduce operations, avoid contamination, and increase the accuracy of the reaction.
(5) Finally, the reaction template is added, and the reaction tube is tightly closed after adding.
(6) Setting up negative and positive controls and blank controls during operation can verify the reliability of the PCR reaction and also assist in judging the credibility of the amplification system.
(7) Use replaceable or high-pressure processable sample injectors as much as possible. Since the sampler is most susceptible to contamination by product aerosols or sample DNA, it is best to use replaceable or high-pressure sample injectors. If there is no such special sampler, at least the sampler should be dedicated during the PCR operation and cannot be cross-used, especially the sampler used for PCR product analysis cannot be used in the other two areas.
(8) Repeat the experiment, verify the results, and draw conclusions carefully.