Finish your experiment strong: What to know about PCR plate sealing

Almost four decades after the original PCR approach was first developed [1], it continues to serve in routine and new applications—including rapid testing for COVID-19.

Despite its familiarity and accessibility, setting up PCR reactions is not a trivial exercise. This is especially true when there are many conditions to assess. To run a PCR experiment, researchers first need to select and acquire each component: primers, nucleotides, buffers, enzymes, and sufficient nucleic acid template. Because these components are tailored to each experiment, selecting PCR ingredients, and accurately setting up the reactions takes time. With this in mind, we can all appreciate the need for diligence during the final step of PCR set-up: sealing plates full of reactions.

Appropriately sealing a PCR plate will help protect your experiment by preventing differential evaporation of samples while in the thermocycler. As water escapes, it alters the pH of the solution and impacts enzymatic function, which can disrupt the accuracy of PCR results. For example, this can alter the cycle threshold (Ct) used to quantify nucleic acids of interest in qPCR, leading to greater variability and reduced reproducibility. To avoid this, consider the following key decision points and tips for picking the right seal for your project.
 

The first step is considering your experimental format. For qPCR experiments that involve making highly sensitive fluorescence measurements directly in the plate, users must select either ultra-clear caps or optical seals to permit maximum fluorescence transmission from the wells. Not all clear seals have enough optical clarity for qPCR, so make sure that you are using optical seals to ensure effective data collection.

While not compatible with qPCR, aluminum foil films provide additional protection for light-sensitive samples. Furthermore, they are often better suited for colder temperature storage (≤–80°C). As a result, foil films are typically your best bet when long term storage is important. Foil seals can also be pierced, which means samples can be transferred from the plate using needles—either manually or with a robotic sampling platform.
 

The next key decision is whether to use plate caps or films. The primary benefit of caps is that they can be used to form tight seals on the plate that can be removed and readily re-sealed as needed. For this to work, they need to fit the specific plate wells and the thermocycler. As such, it’s important to select caps that are compatible with the plate manufacturer and to choose either flat or domed caps, depending on the thermocycler. Since caps can be used on plates as well as PCR tubes, labs can purchase just one kind of cap to match both.

When re-sealing, take care to position the cap on the same well to prevent sample cross-contamination. Applying caps can be tedious for larger experiments where the entire plate is used or when multiple plates are required, though cap installing tools can make this easier.

Film plate seals are more versatile since they can be used on any PCR plate, regardless of the manufacturer. This allows researchers to use film seals on any traditional 96-well PCR plate as well as other plate formats, such as 384- and 24-well plates. Since users can cut the films to match the plate size, they can tailor film seals to their purpose. On the flip side, forming a tight seal with films can be less obvious than caps, since the caps click into place when tightly sealed.

For help sealing your plates with either caps or adhesive films, watch "Learn how to seal a Thermo Scientific PCR Plate" video below.

If film seals are the best fit for your PCR experiment, it’s also important to consider whether you need adhesive or heat films. Generally, adhesive films might be the most straight forward option for labs, since they only require a user to apply the sticky side to a plate surface and carefully establish tight seals, often using adhesive film applicator tools.

On the other hand, heat sealing films generally require the use of plate sealers. While heat sealing adds additional overhead cost due to purchasing a plate sealer, they are superior for long term storage since they provide longer-lasting uniform seals that reduce sample evaporation compared with adhesives.
 

Making a mistake or picking the wrong option when sealing reactions feels a lot like dropping a tray of freshly prepared cookie batter as you make your way to the oven (cue tears). Let’s avoid that heartbreak by carefully considering plate seal options and matching them to the experiment at hand!

Looking for more information to help you find the right PCR plastics for your experiments? Check out our School of Molecular Biology PCR plastics page.
 

1. Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG. Studies on polynucleotides. XCVI. Repair replication of short synthetic DNA's as catalysed by DNA polymerases. J Mol Biol. 1971;56:341–361.