When performing PCR operations, operators should strictly follow some operating procedures to minimize possible PCR contamination or eliminate contamination.
1. Division of operation area: At present, ordinary PCR can not achieve single-person single tube, to achieve complete closed-tube operation, but whether it can achieve single-person single-tube, the experimental operation is required to be carried out in three different areas, PCR Pre- and post-processing should be performed in different isolation zones:
(1) Specimen processing area, including preparation of an amplification template.
(2) Amplification zone, including preparation of the reaction solution and PCR amplification.
(3) Product analysis area, gel electrophoresis analysis, product photographing and preparation of recombinant clones.
(4) Each work area must have a certain degree of isolation, and the operation equipment should be used exclusively. It must have a certain direction. Such as: specimen preparation → PCR amplification → product analysis → product processing.
Remember: Do not get the Other two work areas for the products and equipment in the product analysis area.
2. Packing reagents: The reagents required for PCR amplification should be prepared and dispensed on a clean bench or a vacuum bench equipped with UV lamps. All samplers and tips need to be fixed in place and cannot be used to extract amplified DNA and DNA from other sources:
(1) PCR water should be high pressure double distilled water.
(2) Primers and dNTPs were prepared in a non-PCR amplified product region using high pressure double distilled water.
(3) Primers and dNTPs should be stored separately, and the time should be indicated when dispensing, in case of contamination.
3. Note on experimental operation: Although the residual contamination of the amplified sequence is mostly the cause of the false positive reaction, cross-contamination between samples is also one of the causes. Therefore, it is prudent not only to carry out the amplification reaction, but also to pay attention to all aspects of sample collection, extraction and amplification:
(1) Wear disposable gloves. If you accidentally splash the reaction solution, replace the gloves immediately.
(2) Use disposable tips. Do not mix with the tips of the PCR product analysis room. Do not expose the tips to the air for a long time to avoid aerosol contamination.
(3) Avoid splashing of the reaction solution. To avoid this situation when opening the reaction tube, collect the liquid at the bottom of the tube by centrifuging slightly before opening the lid. If you accidentally splash on your gloves or table, immediately change gloves and wipe the table with dilute acid.
(4) When operating multiple samples, prepare the reaction mixture, first mix the dNTP, buffer, primer and enzyme, and then dispense, so that the operation can be reduced, pollution can be avoided, and the accuracy of the reaction can be increased.
(5) Finally, the reaction template is added, and the reaction tube is tightly closed after the addition.
(6) Establishing a negative positive control and a blank control during operation can verify the reliability of the PCR reaction and assist in judging the credibility of the amplification system.
(7) Use a replaceable or high-pressure sampler as much as possible. Since the sampler is most susceptible to contamination by product aerosol or specimen DNA, it is best to use a replaceable or high-pressure sampler. In the absence of such a special sampler, at least the sampler should be dedicated during the PCR operation and cannot be used interchangeably. In particular, the sampler used for PCR product analysis cannot obtain the other two zones.
(8) Repeat the experiment, verify the results, and carefully conclude.
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