Problem |
Possible Causes |
Solution |
No PCR product is observed |
A PCR component may be missing or degraded. |
A positive control should always be run to insure components are functioning. A checklist is also recommended when assembling reactions. |
There may be too few cycles performed. |
Increase the number of cycles (3-5 additional cycles at a time). |
The annealing temperature may be too high. |
Decrease the annealing temperature in 2-4°C increments. |
The primers may not be designed optimally. |
Confirm the accuracy of the sequence information. If the primers are less than 27 nucleotides long, try to lengthen the primer to 27-33 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primer with a GC content of 45-60%. |
There may not be enough template. |
After increasing the number of cycles has shown no success, repeat the reaction with a higher concentration of template. |
The template may be of poor quality. |
Evaluate the template integrity by agarose gel electrophoresis. It may be necessary to repurify template using methods that minimize shearing and nicking. |
The denaturation temperature may be too high or too low. |
Optimize the denaturation temperature by increasing or decreasing the temperature in 1 °C increments. |
The denaturation time may be too long or too short. |
Optimize the denaturation time by increasing or decreasing it in 10 second increments. |
The extension time may be too short. |
Increase the extension time in 2 minute increments, especially for long templates. |
The reaction may not have enough enzyme. |
0.05 units/µL is sufficient for most applications. It is recommended that the cycling parameters be optimized before the enzyme concentration is increased. In rare cases, the yields can be improved by increasing the enzyme concentration. However, if the enzyme amount is above 0.10 units/µL, higher background levels may be seen. |
Mg++ levels may be suboptimal. |
This is unlikely if the 10X reaction buffer (with MgCl2) is used and the deoxynucleotides do not exceed a concentration of 0.6 mM each (as deoxynucleotide triphosphates can bind Mg++). Typically, MgCl2 is optimized between 1 to 5 mM. Also, EDTA present in the sample at greater than 5 mM will reduce the effective concentration of magnesium. |
Deoxynucleotide amounts are too low. |
This is unlikely if the final concentration of each deoxynucleotide is 0.5 mM. This concentration of dNTPs is suitable for a wide range of applications. If the dNTPs are being prepared in the laboratory, be sure that the final concentration of each deoxynucleotide is 0.5 mM. If the concentration of dNTPs is increased, the Mg++ concentration will need to be increased proportionately. |
Target template is difficult. |
In most cases, inherently difficult targets are due to unusually high GC content and /or secondary structure. Betaine has been reported to help amplification of high GC content templates at a concentration of 0.8-1.3 M. In some cases, the addition of 1-4% DMSO may help. |
The PCR product floated out of the well during direct loading into the gel. |
Use the recommended amount of REDAccuTaq LA DNA Polymerase (2.5 µL per 50 µL reaction) to provide enough glycerol for proper loading of PCR product directly into the gel. |
There may be too many cycles performed. |
By reducing the cycle number, the nonspecific bands may be eliminated. |
The annealing temperature may be too low. |
Increase the annealing/extension temperature in increments of 2-3 °C. |
The primers may not be designed optimally. |
Confirm the accuracy of the sequence information. If the primers are less than 27 nucleotides long, try to lengthen the primers to 27-33 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primers with a GC content of 45-60%. |
Touchdown PCR may be needed. |
“Touchdown” PCR significantly improves the specificity of many PCR reactions in various applications. Touchdown PCR involves using an annealing/extension temperature that is higher than the Tm of the primers during the initial PCR cycles. The annealing/extension temperature is then reduced to the primer Tm for the remaining PCR cycles. The change can be performed in a single step or in increments over several cycles.5 |
Too many cycles may have been performed. |
Reduce the cycle number in 3-5 cycle increments. |
The denaturation temperature may be too low. |
Increase the denaturation temperature in 1 °C increments. |
The extension time may be too long. |
Decrease the extension time in 1-2 minute increments |
Touchdown PCR may be needed. |
See recommendations under “Multiple Products” for procedure. |
There may be too much enzyme in the reaction mix. |
0.05 units/µL is sufficient for most applications. However, this concentration may be too high for some applications. It is recommended the cycling parameters be optimized first, as described above, then, reduce the enzyme concentration to 0.5-0.2X. |
Magnesium concentration may be too high. |
The MgCl2 concentration should be optimized. Typically, the concentration of MgCl2 is optimal between 1 and 5 mM. If the concentration of the dNTPs is 0.5 mM, it is very unlikely that the magnesium concentration is too high. |
The template concentration may be too high. |
Reduce the concentration of the template in the PCR reaction. |
The primers may not be designed optimally. |
See recommendations under “Multiple Products”. |
The extension time may be too short. |
Increase the extension times in 2 minute increments or use touchdown PCR. |
The template concentration may be too low. |
Add additional template in 50 ng increments for genomic DNA or 1-2 ng for viral DNA. |
There may be too few cycles performed. |
Increase the cycle number in 3-5 cycle increments |
The extension time may be too short. |
Increase the extension times in 2 minute increments |
A co-solvent may be required |
Add dimethyl sulfoxide (1-4%) or 0.8-1.3 M betaine final concentration. |