Methods to Reduce Pollution When Doing PCR
How can we effectively reduce pollution when doing PCR?
I. Wear the most suitable laboratory gloves
A major source of contamination in PCR experiments is actually the fact that laboratory gloves often touch the PCR nozzle. Even if you feel like you haven't touched it, you still provide opportunities for pollutants, especially when the experimental gloves do not fit well, which makes the experimental operation not very dexterous and easy to cause pollution.
II. Wipe the workbench with 70% ethanol before the PCR experiment
The nuclease contained in our dander will affect the result of the PCR reaction. Nucleases such as DNase can cut DNA and affect the results of experiments. Especially when there is no band, it may be contaminated by nuclease. In order to avoid such nuclease contamination and other bacterial contamination, it is recommended that you disinfect and wipe the counter-tops and laboratory gloves with 70% ethanol before the experiment.
III. Hold the middle and lower part of the PCR tube when adding reagents
When you start the experiment, you come across a lot of equipment, even if you wipe it with ethanol, the experiment gloves are still not clean. In this case, it is necessary to keep the experimental gloves as far away as possible from the PCR tube opening to prevent contaminants from entering it. Although it may seem trivial, it can often directly affect the results of PCR.
VI. Separate the reagents (aliquot)
Before each experiment, it is better to separately put double deionised water into 1ml or 1.5ml eppendorf tubes. In this way, throw it away every time it is used to ensure the quality of deionized water. In addition, it is recommended that you also separate other reagents, such as buffers, polymerases, dNTPs, primers and so on. This can avoid contamination of the reagents in the original bottles due to repeated use.
V. Close the lid of the PCR tube carefully
Although it looks very inconspicuous, we often waste valuable experimental materials and time just because the lid is closed hastily. If the lid is closed too hastily, especially when using a PCR strip, it is likely that the mixture inside will adhere to the top of the tube wall or spill. And if you cover it too quickly without careful inspection, it is very likely that the cover is not tight, causing the water to evaporate in the PCR machine. This will result in high concentrations of dNTPs and inhibit the PCR reaction. Even if there are results in subsequent the gel-running, it is difficult to guarantee the reliability and repeatability of the data.
VI. Separate reagents
PCR reagents, products and other molecular biology reagents should be placed separately. For example, be sure to store PCR tubes separately in packaging bags. And if conditions permit, the mixture before and after the PCR reaction should be placed in different places in the refrigerator.