A person wearing gloves pipetting liquid into a PCR strip tube

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 causeRecommendation
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 causeRecommendation
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 causeRecommendation
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 causeRecommendation
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 causeRecommendation
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.
  • Find a PCR tube compatible with your thermal cycler with a PCR and qPCR Plastics Selection Tool
High pressure exerted by the thermal cycler lid
  • 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 causeRecommendation
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 causeRecommendation
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 causeRecommendation
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 installing tool if available (see the video on tool usage).

Film not adhering to PCR plate

Possible causeRecommendation
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 causeRecommendation
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 installing 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.


PCR plastics how-to videos

For Research Use Only. Not for use in diagnostic procedures.