Is separating Pre-PCR and Post-PCR working areas necessary?

PCR is extremely sensitive! The Polymerase Chain Reaction (PCR) amplifies minuscule amounts of DNA. Therefore, even a trace of amplified DNA (from a previous experiment) can contaminate new samples or reagents used in the assay, thus falsifying results. To learn more about PCR, see our RNA Lexicon chapter on RNA-Seq Library Preparation.

We recommend you have two spaces (areas) for pre- and post-PCR work!

• Pre-PCR: In this area precious samples are handled prior to amplification. Keeping it free of "contaminating" amplified DNA is key for reliable and correct results.

• Post-PCR: Once DNA is amplified, contamination risk to the current experiment is no longer a threat. In the post-PCR area, the sample is amplified and the resulting DNA library is analyzed, e.g., by electrophoresis, Fragment Analyzer, NanoDrop, and further prepared for subsequent sequencing.

Separation can be achieved through:

• Two rooms: Ideally, pre-PCR and post-PCR labs are situated in dedicated rooms that can be closed individually.

• Dedicated equipment: Use separate instruments (e.g., PCR cyclers, fridges, freezers, centrifuges), equipment (e.g., pipettes, racks, magnetic separators), and reagents in each area.

• Airflow control: Slightly positive air pressure in pre-PCR will prevent contamination from the post-PCR area.

IMPORTANT: Never move samples, equipment, or reagents from post-PCR to the clean pre-PCR area without thorough decontamination. Always change gloves and ideally protective equipment when moving between areas.