Use a unidirectional workflow during experiment setup. Separating pre- and post-amplification areas is key to preventing contamination. Prepare your PCR master mix in a template-free room (see next bullet) using reagents that never come into contact with potential sources of contamination. Maintain a separate area for analyzing PCR amplicons.

Maintain a clean room for preparing master mixes. Prepare your PCR master mix in one room, and then add your template in a separate room to avoid introducing template into the clean room. Prepare components using a benchtop hood when possible to minimize risk of contamination; a benchtop hood is also simple to decontaminate if needed. Keep enzyme mixes, water, primers and probes, pipettes, tubes, tips, and plates in a room where template is not isolated or stored.

Regularly decontaminate equipment. Clean pipettes and other non-porous surfaces with 5% bleach solution to degrade any DNA that may be present. It is easiest to use a spray bottle for this purpose. Leave the solution on the surface for a few minutes to ensure complete degradation of DNA. Alternatively, you can use UV sterilization to decontaminate equipment, including tubes, racks, and pipettes.

Use positive displacement or filter tips for reaction setup. Pipettes are a common source of contamination by aerosols. Filter tips provide a barrier between the pipettes and the liquid being measured, preventing the transfer of aerosols into samples and reagents. Positive displacement pipettes have no air interface between the piston and reagents being measured and, therefore, limit the risk of aerosol contamination.