Using Micro-plates and Sealing Films in qPCR and PCR Experiments

Polymerase chain reaction (PCR) and quantitative PCR (qPCR) are fundamental techniques in molecular biology, used for amplifying and quantifying DNA. The success of these experiments depends not only on the reagents and thermal cycler conditions but also on the quality of labware—particularly microplates and sealing films. These essential consumables impact reaction efficiency, reproducibility, and contamination control.

Microplates in PCR and qPCR

Microplates, also called PCR plates, are available in various formats, including 96-well and 384-well plates, designed to fit thermal cyclers and real-time PCR instruments. The choice of microplate depends on factors such as sample volume, throughput, and compatibility with specific instruments.

Types of Microplates

1. Well Format:

• 96-well plates: Standard for most PCR and qPCR applications, balancing throughput and reagent volume.

• 384-well plates: Ideal for high-throughput assays, allowing more reactions in a single run while using smaller volumes.

2. Material and Transparency:

• Polypropylene plates: Provide chemical resistance and low binding to biomolecules, ensuring sample integrity.

• Clear, white, or frosted plates: White plates enhance fluorescence signal in qPCR, while clear plates are useful for endpoint PCR visualization.

3. Well Shape and Design:

• V-bottom or U-bottom wells: Aid in efficient sample retrieval.

• Low-profile plates: Minimize dead volume and improve heat transfer, reducing reaction time.

Sealing Films for PCR and qPCR

Proper sealing of microplates is critical to prevent sample evaporation, cross-contamination, and inconsistent results. Sealing films and foils come in various types, each suited for different applications.

Types of Sealing Films and Their Applications

1. Optically Clear Films:

• Ideal for qPCR as they allow real-time fluorescence detection.

• Minimize signal interference for accurate quantification.

2. Adhesive vs. Heat-Sealing Films:

• Adhesive films: Easy to apply, suitable for standard PCR but may not withstand prolonged high temperatures.

• Heat-sealing films: Provide a stronger, more consistent seal, ideal for long qPCR runs and high-throughput applications.

3. Aluminum Foil Seals:

• Excellent for PCR when optical clarity is not required.

• Effective in preventing evaporation and contamination in endpoint PCR.

4. Pierceable Films:

• Allow automated pipetting without removing the seal.

• Useful in high-throughput screening applications.

Best Practices for Using Microplates and Sealing Films

1. Choose the Right Plate and Seal for Your Experiment:

• Match the microplate type to your instrument and experiment needs.

• Use optically clear films for qPCR to ensure accurate fluorescence readings.

2. Ensure Proper Sealing:

• Apply consistent pressure when sealing plates to prevent uneven coverage.

• For adhesive seals, avoid excessive handling to prevent contamination.

3. Reduce Evaporation and Well-to-Well Contamination:

• Use low-evaporation films for long-run qPCR experiments.

• Properly seal plates before centrifugation or vortexing to prevent sample loss.

4. Verify Compatibility with Your Thermal Cycler:

• Ensure the selected microplate fits the block design of your thermal cycler.

• Confirm that the sealing film does not interfere with instrument optics in qPCR.

Conclusion

The choice of microplates and sealing films in PCR and qPCR experiments significantly affects data accuracy, efficiency, and reproducibility. Selecting the right labware and following best practices ensures optimal reaction performance while minimizing errors and contamination. By investing in high-quality microplates and appropriate sealing films, researchers can improve the reliability of their molecular biology experiments and achieve consistent, reproducible results.