A PCR plate is a tray of small wells used to hold reaction mixtures for the Polymerase Chain Reaction (PCR). Sealing the plate correctly is essential for ensuring reliable genetic analysis. The primary goal of a proper seal is to maintain the precise volume and concentration of reagents throughout the rapid heating and cooling cycles of the thermal cycler. Without an effective barrier, minimal sample evaporation can concentrate reactants, potentially invalidating quantitative results. A secure seal also prevents the transfer of aerosols or liquid droplets between neighboring wells, which would lead to cross-contamination.
Choosing the Right Sealing Material
Selecting the appropriate sealing material directly impacts the success of the experiment and the integrity of the data. Adhesive films, often made from clear polypropylene or polyester, are generally suitable for standard endpoint PCR applications. These films offer good optical clarity and are robust enough to withstand thermal cycling temperatures, typically up to 100°C. Aluminum foils are completely opaque and are preferred when light sensitivity is not a concern, such as in post-PCR analysis or for long-term sample storage.
For quantitative PCR (qPCR), fluorescent signals are measured through the plate lid, requiring a highly specialized optical adhesive film. These films are engineered to transmit light efficiently across the necessary excitation and emission wavelengths without causing signal interference or autofluorescence. Using a standard, non-optical film or opaque foil in a qPCR assay will block the light path, making accurate quantification impossible. The optical properties of the chosen film must be validated for compatibility with the instrument’s detection system.
While manual sealing is common, high-throughput laboratories often utilize heat-sealing methods for superior uniformity and bond strength. Heat seals use an aluminum or polyester foil activated by a dedicated heat-sealing machine, creating a permanent, solvent-resistant barrier suitable for automated systems and extended storage. Alternative sealing methods like cap strips or individual well caps are only appropriate for tube strips or specific plate types, not the standard 96- or 384-well plates that rely on adhesive seals. The material choice should prioritize temperature stability and chemical resistance to the reagents.
Step-by-Step Manual Sealing Techniques
Manually applying an adhesive seal begins with meticulous plate preparation to ensure maximum adhesion. The entire rim surface of the PCR plate must be completely clean and dry before the sealing film is applied. Any residual liquid, buffer, or fingerprints on the plastic rim will compromise the seal’s integrity and lead to failure during thermal cycling. This preparatory step provides the foundation for a reliable seal.
Once the plate is ready, carefully peel the backing from the sealing film and align it precisely over the plate wells. Proper alignment ensures the adhesive material covers the entire top surface without overlapping the sides or leaving any well edges exposed. Gently touch the center of the film to the plate surface, then lightly press down on the two opposite short edges to “tack” the film in place. This initial tacking stabilizes the film for the full application.
The next step involves using a specialized rubber roller or a flat-edged squeegee tool to firmly apply pressure across the entire surface of the film. Begin rolling from the center of the plate and move outward toward the edges, applying consistent and firm downward force. This action eliminates trapped air pockets and forces the adhesive into intimate contact with the plastic surface, especially around the individual well rims.
Pay close attention to the areas between the wells, where the plastic surface is slightly raised, as these points form the primary seal against evaporation. Rolling over these inter-well spaces multiple times helps ensure a uniform bond across the entire array of reactions. Failing to adequately press down the adhesive in these regions is a common cause of sample loss during temperature cycling.
Finally, secure the entire perimeter of the sealing film by pressing down firmly on all four outer edges with the squeegee or roller edge. This focused pressure prevents the film’s edges from lifting during the high-temperature phases of the PCR cycle, a common failure point. A well-secured perimeter maintains the overall tension and integrity of the seal throughout the thermal program.
Verifying the Seal and Preventing Common Errors
After manual application, the sealed plate requires a thorough quality check before being placed in the thermal cycler. Visually inspect the film for wrinkles, creases, or air bubbles that might span across a well opening or the space between wells. A perfectly sealed plate will appear taut and smooth, with the adhesive material bonded to the entire plate rim and the plastic surrounding each well. Evidence of poor contact suggests a potential failure point.
A brief, low-speed centrifugation step is recommended for every sealed plate immediately after the film is applied. This spin-down forces liquid droplets clinging to the well walls or the underside of the seal to the bottom of the reaction vessel, ensuring accurate starting volumes. If the seal is compromised, this centrifugation often reveals the failure by showing liquid leakage or cross-contamination before the run begins, allowing for correction.
Evaporation, a frequent failure, is often traceable to insufficient pressure applied during sealing, particularly at the plate’s edges or over the inter-well dividers. If the thermal cycler’s heated lid is not applying even pressure, the plate’s thin plastic can deform slightly, leading to small gaps in the seal during the high-temperature phase. Condensation under the film, while sometimes unavoidable, can be minimized by ensuring the thermal cycler’s heated lid is set to a temperature slightly higher than the maximum cycling temperature to prevent vapor accumulation.