PCR/qPCR Plastics Troubleshooting Guide

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:

On this page

  1. PCR/qPCR issues (related to plastics)
    1. No or low amplification
    2. Low qPCR signal
    3. Variable qPCR data
    4. False positive results
  1. Tubes and plates issues
    1. Suboptimal fit to the thermal block
    2. Deformation or crush
    3. Melting
    4. Sticking to the block following PCR
    5. Chemical leaching
  1. Sealing issues
    1. Sample loss/evaporation
    2. Adhesive film not sticking
    3. Cap popping

1. PCR/qPCR issues (related to plastics)

a. No or low amplification

Possible cause Recommendation
Suboptimal fit to the thermal cycler’s block
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 wells.
  • In fast PCR/qPCR, ultrathin-walled and low-profile plastics are recommended for robust thermal conductivity and reduced evaporation.
Presence of inhibitors or nucleases
  • Verify that the 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 inhibitors and nucleases.
Overfilled or underfilled
  • Do not use more than the recommended fill volume of the tube or well, to enable optimal heat transfer.
  • Avoid underfilling, to prevent sample evaporation that could impact reaction efficiency.

b. Low qPCR signal

Possible cause Recommendation
Non-optimal well color
Non-optimal seal clarity
  • Ensure that sealing films and caps are optically clear or ultraclear to minimize distortion of fluorescence signals.

c. Variable qPCR data

Possible cause Recommendation
Well-to-well variation
  • Select plastics with white wells instead of clear wells, since white-wall plastics help improve well-to-well consistency by preventing signal refraction out of the wells.
Suboptimal seal clarity

d. False positive results

Possible cause Recommendation
Presence of DNA contaminants
  • Verify that the plastics and seals are manufactured in a clean-room environment with stringent guidelines to prevent DNA contamination.
  • Request a Certificate of Analysis from the manufacturer, for proof that the production lot has been tested for the presence of DNA contaminants.
  • For sensitive applications like qPCR for human identification, ensure that sealing films have been treated with ethylene oxide by the manufacturer (per ISO 18385 requirements) to destroy potential DNA contaminants.
Improper sealing
  • Ensure that all wells are sealed properly to avoid cross-contamination.
  • If using tube strips, consider using ones with attached caps to enable opening and closing of each tube independently and help prevent cross-contamination.

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2. Tubes and plates issues

a. Suboptimal fit to the thermal block

Possible cause Recommendation
Incompatible design
  • Choose PCR/qPCR plastics that have been verified to be compatible with the thermal cycler being used, for optimal fit.
  • Consult plastics selection guides from the manufacturer for optimal fit to the thermal cyclers being used.

b. Deformation or crush

Possible cause Recommendation
Incompatible design
  • Choose PCR/qPCR plastics that have been verified to be compatible with the thermal cycler being used, for optimal fit.
  • Consult plastics selection guides from the manufacturer for optimal fit to the thermal cyclers being used. When appropriate, choose skirted plates for extra support and strength.
  • For use with robotic handling, select skirted plates with a polycarbonate (hard shell) frame to withstand forces exerted by the grippers.
High pressure exerted by the thermal cycler lid
  • When tubes or tube strips are used, distribute them evenly across the block to balance the exerted pressure. If necessary, place empty tubes in the block to distribute the lid pressure.
  • For certain thermal cyclers, consult the manufacturer’s guide to use of accessories (e.g., trays and retainers) to avoid crushing tubes and tube strips (see the video on tray and retainer usage).
  • Switch to domed caps from flat caps, or vice versa. While domed caps may better withstand the pressure from the thermal cycler lid, flat caps take up less space between the tubes and the lid.
  • If it is adjustable, do not overheat (usually 5°C above sample temperature is sufficient) or overtighten the thermal cycler lid.
  • Verify the integrity and function of the thermal cycler lid to ensure its spring or closing mechanism is functioning correctly.

c. Melting

Possible cause Recommendation
Suboptimal material
  • Ensure that the plastics are made of polypropylene, which can withstand thermal cycling. Use plastics from a different production lot, if needed, and contact the manufacturer if the issue persists.

d. Sticking to the block following PCR

Possible cause Recommendation
Rapid heating and cooling cycles

e. Chemical leaching

Possible cause Recommendation
Suboptimal manufacturing process
  • Consider PCR/qPCR plastics made of medical-grade or 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.

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3. Sealing issues

a. Sample loss/evaporation

Possible cause Recommendation
Improper sealing Films:
  • 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 video on tool usage).
  • If compatible, choose plates with a flat deck (instead of raised deck) to facilitate sealing. For automatic sealing, use of plates with engraved lettering (instead of raised lettering) may help ensure sealing.
Caps:
  • 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).

b. Adhesive film not sticking

Possible cause Recommendation
Nonsticky, pressure-sensitive films
  • Apply sufficient firm pressure to establish complete contact and strong bonding with the well rims. Use an adhesive seal applicator if available (see the video on tool usage).
  • Alternatively, consider convenient cap mats that exhibit low sample evaporation.

c. Cap popping

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 if available (see the video on tool usage).
  • Verify that the shape of the caps being used (flat or domed) provides optimal fit with the thermal cycler’s lid.

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For more troubleshooting tips, please review the PCR and qPCR troubleshooting guides, visit our Support Center, or contact our technical support team.

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