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Troubleshooting PCR and RT-PCR Amplification

Many of the common problems with PCR and RT-PCR are identified during agarose gel electrophoresis of the reaction products. These include the absence of the expected amplification product, the presence of nonspecific products, excessive smearing, and the presence of a “primer dimer” band. A summary of the possible causes and solutions for these problems is provided below.

ProblemPossible causeSolution
No bands on gelThe thermal cycler was not functioning properlyIf a positive control was not prepared, consider testing reagents in a control reaction with template and primers previously shown to function properly.
Insufficient template or too much template was addedTitrate the amount of template required for your system.
For DNA: Template quality was poor or contains inhibitorsImpure DNA can fail to amplify properly. Use freshly prepared DNA or isolate template by another method.
Primer concentration was too low or was unbalancedMake sure primer concentration is within recommended range and that concentration of both PCR primers is the same.
Nucleases were introducedPrepare new template. Be sure to follow precautions against introducing nucleases. This is particularly important when the starting material is RNA.
For RNA: Template is in buffer containing EDTAEDTA present in the RNA solution will chelate Mg2+ required for the RT-PCR and may lead to suboptimal results. To compensate, add additional Mg2+ when assembling the reactions. Refer to product instructions for details.
Reagents were not thawed properly before useThaw and thoroughly mix all reagents before setting up reactions.
Primer annealing temperature was too highDecrease annealing temperature in 2 °C increments.
Denaturing temperature was suboptimalTry increasing the temperature or duration time of the initial denaturation step. This is especially useful for GC rich templates.
Extension time was too shortIncrease extension times. This can be especially important for long PCR.
Thermal cycler was not at correct temperature
Repair/calibrate the thermal cycler or use a different machine.
Extra, nonspecific bands on gel
Primers hybridized to a secondary site on the templateDesign new primers that are less specific for the secondary site. Increase the annealing temperature by increments of 2 °C to 5 °C.
DNA contamination was introduced in primers or buffersRun a negative control reaction (no template). Prepare fresh materials if contamination is detected.
For RNA: Template is contaminated with DNAPerform a DNase step and completely inactivate/ remove DNase. Alternatively, prepare RNA using a different method.
Too much template was addedTitrate the amount of template for your system.
Primer concentration was too highTitrate the amount of primers for your system.
Thermal cycler was not at correct temperature
Repair/calibrate the thermal cycler or use a different machine.
Excessive smearing of amplified DNA
Too much template was addedTitrate the amount of template for your system.
Too many cycles were performedReduce the number of cycles below 35.
Thermal cycling conditions were not optimal for your thermal cyclerOptimize conditions based on manufacturer’s recommendations.
The annealing temperature was too lowWhen using Ready-To-Go Beads, re-optimization of the annealing temperature might be required. Increase the annealing temperature by increments of 2 °C to 5 °C.
Primer concentration was too highTitrate the amount of primers for your system.
Extension time was too longDecrease extension time in small increments.
For DNA: Template quality was lowImpure DNA can fail to amplify properly. Use freshly prepared DNA or isolate template by another method.
Too much DNA was loaded on gelRe-run with less sample.
Primer dimers visible
Too much primer was added/ primer concentration too highTitrate the amount of primers for your system.
Primers possess complementary overlapping sequenceDesign primers to avoid self-complementary internal sequences.
Increase annealing temperature. Use illustra™ Hot Start Mix PCR products.
Table 7.4.Possible causes and solutions for problems with PCR and RT-PCR amplification.
Materials
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