PCR room contamination monitoring and defense
2012 National Justice and Blood Center International Standard DNA Laboratory Observation will be held in June next year in Qingdao International Convention and Exhibition Center, the Observation will be adhering to the concept of creating the world's top exhibition, will show the world-class level, in line with international standards of DNA laboratory prototype room. And will be in the process of displaying to the participants of the free distribution of laboratory solutions, this article is a few days ago by the organizing committee to show part of the series of solutions.
The biggest attraction of PCR reaction is its amplification power and high sensitivity, but easy contamination has always been a big problem for laboratories, and a very small amount of contamination may cause a false-positive reaction, destroying the test results. How to monitor and prevent PCR contamination is now an important issue for laboratories.
I. Pollution monitoring
A good PCR laboratory must always pay attention to the monitoring of contamination, consider whether the contamination, such as contamination, what is the cause. Through effective monitoring, take the right measures to prevent or eliminate contamination.
Control tests are an important means of monitoring PCR contamination.
1, negative control: negative control is necessary for each PCR experiment. It includes a specimen control and a reagent control, which confirms whether the sample and reagent are infected.
2. Positive control: It is an important reference mark for the success of the PCR reaction and whether the location and size of the product bands meet the theoretical requirements. Positive control should choose the amplification degree of medium, good reproducibility, identified as the product of the specimen. However, positive controls have a high probability of contaminating the test or amplification sample. Thus, when a PCR reagent has been used to stabilize, the inspector completely mastered, in the subsequent experiments should be as far as possible to avoid setting up a positive control. To reduce the probability of contamination.
3, select different regions of the primers for PCR amplification.
4. Repeatability test.
Two, to prevent contamination of the method
1, a reasonable separation of the laboratory: the processing of samples, preparation of PCR reaction solution, PCR cycle amplification and identification of PCR products, such as steps for partitioning or sub-office, in particular, the sample processing and identification of PCR products and other steps are strictly separated. The ideal partition is the specimen processing area, the PCR reaction solution preparation area, the PCR cycle amplification area, and the PCR product identification area.
2, PCR reagents to do a good job of pre-mixing and partitioning: all PCR reagents should be partitioned in small quantities, PCR reaction solution should be pre-prepared as far as possible, and then partitioned in small quantities, stored at -20 ℃ to reduce the number of repeated spiking to avoid the chance of contamination. In addition, PCR reagents and PCR reaction solution should be stored separately from the samples and PCR products, and should not be put in the same ice box or refrigerator.
3. Pipette gun contamination protection: The pipette gun is a device that is very susceptible to contamination. If the sample or template nucleic acid is inadvertently sucked into the gun or stick on the gun head, it will become a serious source of pollution, so be extra careful when using the gun, suck the sample slowly, and try to complete a one-time, avoid multiple suction, to avoid cross-contamination or aerosol contamination.
4, reduce the number of PCR cycles: PCR products to reach the detection level is appropriate.
5, to prevent operator contamination: the use of disposable gloves, pipette tips, small centrifuge tube.