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:
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.
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
Ensure that sealing films and caps are optically clear or ultraclear to minimize distortion of fluorescence signals.
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.
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.
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.
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
Carefully remove stuck tubes and plates from the thermal cycler block without damaging the vessels. Use of a tool designed for plate removal may facilitate the process (see the
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.
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
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
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
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
Verify that the shape of the caps being used (flat or domed) provides optimal fit with the thermal cycler’s lid.
