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.

Table 7.4. Possible causes and solutions for problems with PCR and RT-PCR amplification.

Problem	Possible cause	Solution
No bands on gel	The thermal cycler was not functioning properly	If 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 added	Titrate the amount of template required for your system.
	For DNA: Template quality was poor or contains inhibitors	Impure DNA can fail to amplify properly. Use freshly prepared DNA or isolate template by another method.
	Primer concentration was too low or was unbalanced	Make sure primer concentration is within recommended range and that concentration of both PCR primers is the same.
	Nucleases were introduced	Prepare 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 EDTA	EDTA 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 use	Thaw and thoroughly mix all reagents before setting up reactions.
	Primer annealing temperature was too high	Decrease annealing temperature in 2 °C increments.
	Denaturing temperature was suboptimal	Try increasing the temperature or duration time of the initial denaturation step. This is especially useful for GC rich templates.
	Extension time was too short	Increase 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 template	Design 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 buffers	Run a negative control reaction (no template). Prepare fresh materials if contamination is detected.
	For RNA: Template is contaminated with DNA	Perform a DNase step and completely inactivate/ remove DNase. Alternatively, prepare RNA using a different method.
	Too much template was added	Titrate the amount of template for your system.
	Primer concentration was too high	Titrate 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 added	Titrate the amount of template for your system.
	Too many cycles were performed	Reduce the number of cycles below 35.
	Thermal cycling conditions were not optimal for your thermal cycler	Optimize conditions based on manufacturer’s recommendations.
	The annealing temperature was too low	When 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 high	Titrate the amount of primers for your system.
	Extension time was too long	Decrease extension time in small increments.
	For DNA: Template quality was low	Impure DNA can fail to amplify properly. Use freshly prepared DNA or isolate template by another method.
	Too much DNA was loaded on gel	Re-run with less sample.
Primer dimers visible	Too much primer was added/ primer concentration too high	Titrate the amount of primers for your system.
	Primers possess complementary overlapping sequence	Design primers to avoid self-complementary internal sequences. Increase annealing temperature. Use illustra™ Hot Start Mix PCR products.