When problems arise in PCR and qPCR experiments, you may first seek to troubleshoot reagents and instrument settings (related: troubleshooting guides on PCR and qPCR). However, since the amplification reaction takes place within a plastic vessel, it is also important to look at potential issues related to PCR/qPCR plastic consumables, as suggested below.
1. Poor PCR results that may be caused by plate and tube issues
No or low amplification
Possible cause
Recommendation
Suboptimal fit to the thermal cycler’s block
Choose PCR/qPCR plastics that have been verified to be compatible with the thermal cycler being used, for optimal fit and performance.
Consult a PCR plastics selection guide to find a PCR consumable verified to be compatible with the thermal cycler(s) being used.
Suboptimal material or construction
Ensure that the plastics selected are constructed with characteristics for optimal heat transfer during PCR/qPCR, such as uniform, thin-wall polypropylene wells.
In fast PCR/qPCR, ultrathin-walled and low-profile plastics are recommended for robust thermal conductivity and reduced evaporation.
Contamination by nucleases
Verify that the PCR plastics are manufactured in a clean-room environment with stringent guidelines to prevent dust and nuclease contamination.
Request a Certificate of Analysis from the manufacturer for proof that the production lot has been tested for the presence of nucleases.
Overfilled or underfilled
Do not use more than the recommended fill volume of the tube or plate well, to enable optimal heat transfer.
Avoid underfilling (which leaves a lot of head space in the tube), to prevent sample evaporation during PCR that could impact reaction efficiency.
Low qPCR signal
Possible cause
Recommendation
Loss of signal through well walls
Select plastics with white wells instead of clear wells, since white-well plastics reduce signal refraction and enhance fluorescence reflection to the qPCR detector.
Non-optimal seal clarity
Ensure that sealing films and caps are optically clear or ultraclear to minimize distortion of fluorescence signals.
Variable qPCR data
Possible cause
Recommendation
Well-to-well variation
Select qPCR plates or tubes with white wells instead of clear wells, since white-well plastics help improve well-to-well consistency by preventing crosstalk.
Suboptimal seal clarity
Ensure that sealing films and caps are optically clear or ultraclear to minimize distortion of fluorescence signals.
False positive PCR/qPCR results
Possible cause
Recommendation
Presence of DNA contaminants
Request a Certificate of Analysis from the manufacturer, for proof that the production lot has been tested for the presence of human DNA.
For sensitive applications like qPCR for human identification, ensure that sealing films have been treated with ethylene oxide (per ISO 18385 requirements) to destroy potential DNA contaminants.
Improper sealing
Ensure that all wells are sealed properly to avoid cross-contamination. (See sealing issues section below)
If using tube strips, consider using ones with attached caps to enable opening and closing of each tube independently to help prevent cross-contamination.
2. Physical PCR plate and tube issues
Deformation or crushing of PCR tubes
Possible cause
Recommendation
Incompatible design
Use the cap style recommended by your thermal cycler manufacturer.
Choose PCR/qPCR tubes that have been verified to be compatible with the thermal cycler being used.
Distribute tubes and strips evenly across the block to balance the exerted pressure. If necessary, place empty tubes in the block to distribute the lid pressure.
Consult manufacturer’s guides on use of trays and retainers (see the video on tray and retainer usage).
Make sure the thermal cycler lid is not overtightened.
Verify that the spring or closing mechanism of the thermal cycler lid is functioning correctly.
Plate/tube melting or adhering to the block following PCR program
Possible cause
Recommendation
Suboptimal material
Ensure that the PCR tubes/plate wells are made of high-grade polypropylene, which can withstand thermal cycling. Try a different production lot, if needed, and contact the manufacturer if the issue persists.
Rapid heating and cooling cycles
Carefully remove stuck tubes and plates from the thermal cycler block without damaging the vessels. Use of a plate removal tool may facilitate the process (see the video on tool usage).
Chemical leaching from PCR plasticware
Possible cause
Recommendation
Suboptimal manufacturing process/materials
Consider PCR/qPCR plastics made of molecular biology-grade virgin polypropylene for lot-to-lot consistency and purity.
Choose plastics from trusted and reliable manufacturers that follow stringent manufacturing guidelines and are transparent about their production processes.
3. PCR plate sealing issues
PCR sample loss/evaporation
Possible cause
Recommendation
PCR film not fully sealing the wells
Press the film firmly—along every edge (including the outer edges) and between and around the wells—to seal the well rims properly. Use a sealing applicator if available (see the videos).
If compatible with your thermal cycler, choose plates with a flat deck (instead of raised deck) to facilitate sealing.
For automatic sealing, the use of plates with engraved lettering (instead of raised lettering) may help ensure sealing.
PCR caps not firmly in place
Check compatibility of the cap strips with the tubes and plates being used.
Ensure that the caps are tightly in place. Use a cap sealing tool if available (see the video on tool usage).
Film not adhering to PCR plate
Possible cause
Recommendation
Use of pressure-sensitive films
Apply sufficient firm pressure to establish complete contact and strong bonding with the well rims. Use a seal applicator (see the video on tool usage).
Alternatively, consider convenient cap mats, which can prevent sample evaporation.
PCR cap popping off
Possible cause
Recommendation
Suboptimal fit
Check compatibility of the caps with the tubes and plates being used.
Ensure that the caps are tightly in place. Use a cap sealing tool to apply and tighten the caps (see the video on tool usage).
Verify that the shape of the caps being used (flat or domed) are compatible with the thermal cycler’s lid.
