How to Design Your PCR Lab

PCR (Polymerase chain reaction) is a very sensitive molecular biology technique capable of producing a large number of copies of DNA from a tiny amount of starting material. This makes PCR a basic and efficient experimental method. However, due to its susceptibility to amplicon and sample contamination, special careful consideration is required when planning and designing PCR laboratories.

Laboratory Layout

An ideal PCR lab would consist of two rooms and two areas, each designed for a specific task. The first room should be dedicated to pre-PCR activities and can be further divided into main mixture preparation areas and sample preparation areas. In this area, the air pressure should be kept slightly positive to prevent the inflow of aerosols and ensure a clean environment.

The second room should have a dedicated area for DNA amplification and another area for product analysis. The air pressure in this area should be kept slightly negative to ensure that amplicon aerosols do not escape, thereby avoiding the risk of cross-contamination.

If your space or budget constraints do not allow you to build two rooms, you can set up the pre-PCR area and the amplification and analysis area in the same room, but you must make sure that they are as far away from each other as possible. This is particularly important because the amount of DNA in the pre-PCR stage is usually very low, while the concentration of DNA after amplification is very high. If analyzed in the same space, false positive results may result from amplicon contamination.

One-way Workflow

It is essential to ensure that the workflow of the laboratory is one-way. It is prohibited to bring materials or reagents used in the amplification and analysis area into the pre-PCR space, especially if they have not been thoroughly purified. This means that each area needs to be equipped with dedicated equipment, such as two different sets of pipettes, to reduce the possibility of cross-contamination. In addition, laboratory workers must replace personal protective equipment when switching areas, as these equipment may be contaminated with amplicon aerosols.

Considering time separation is also an effective preventative measure when designing a laboratory. You can perform the PCR reaction in the morning and the amplification and analysis steps in the afternoon. Although this may limit flexibility, it helps prevent contamination problems, thus avoiding the hassle of repeating experiments.

PCR Device Tips

PCR laboratories typically require a variety of equipment, including centrifuges, vortex mixers, pipettes, refrigerators, freezers, thermal cyclers, and electrophoresis systems. Depending on the size of your lab and your specific application, the amount of equipment you need may vary. We do not provide a specific "shopping list" here, but rather a list of things to look for when purchasing equipment and consumables to minimize contamination from PCR reactions.

Critical Equipment

Laminar Flow or Biosafety Cabinets

In view of the possible presence of amplicon aerosols in the pre-PCR space, PCR reactions in laminar flow hoods or biosafety cabinets are recommended. Before and after the operation, clean the workspace with a bleach solution to ensure the hygiene of the environment.
Pipette tips and other consumables

In a PCR setup, the use of filter pipette tip can effectively prevent aerosols from entering and contaminating your pipette, preventing pre-existing aerosols from affecting the accuracy of the main mixture or sample. In order to minimize the consumption of filter headers, it is recommended to fill all tubes with only one set of headers and then treat each sample with a separate header. At the same time, it is recommended to add the sample last, as it is easier to distribute it to the liquid, and this reduces the risk of sample atomization. Learning best pipetting practices can help prevent aerosol formation.

Reagent Storage

Make sure there are enough vials in the lab when the PCR reagent arrives. Subpacking the reagent into smaller containers extends its shelf life and prevents it from going through too much freezing and thawing. In addition, if the reagent is contaminated, you will still have a clean, evenly divided sample, avoiding the loss of total abandonment.

Product Quality Control

Finally, ensure that all consumables and equipment are free of DNase, RNase and PCR inhibitors. Always choose a sterile product from the manufacturer and ensure that the suction heads and consumables they provide do not contain any of these potential contaminants.

Cleaning and Pollution Control

Cleaning and pollution control may not be a priority at the beginning of the lab setup, but when the lab is up and running, cleaning and control will become critical. Before and after setting up, you need to wipe the workspace with a newly formulated bleach solution and distilled water to ensure it is clean. The same cleaning procedure should be performed in the amplification and analysis areas. Regular cleaning of pipettes, equipment, door handles, and refrigerator and freezer handles can significantly reduce the risk of contamination.

Because PCR testing is so sensitive, all the precautions described here may still not fully guarantee that the experiment will never be contaminated. Therefore, it is very necessary to implement appropriate control measures in order to detect pollution in time. Always include negative and positive controls, which will help identify contamination of the main mixture and confirm the performance of extraction protocols, reagents, and amplification steps. In addition, you should also monitor the laboratory's positive rate to ensure that unexpected increases have an identifiable cause, such as seasonal outbreaks.

Conclusion

I hope this article will help you in the process of setting up or perfecting your PCR laboratory. If you are interested in the following workflow and how to determine the concentration and purity of PCR products, please read our related article on DNA quantification.