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Browse our frequently asked technical questions by selecting a product below.
My results contain a high background of non-specific amplicons or smears. What trouble-shooting suggestions do you have?
If smears are a concern, it’s good practice to ensure they are not an artifact of running agarose gel electrophoresis with sub optimal conditions. Sub optimal conditions can include high voltage or not allowing enough time for the gel to set1.
You may also need to troubleshoot the PCR reaction and consider the suggestions below2.
- Primers should be designed to prevent primer-primer interactions and improve specificity.
- Increase the annealing temperature or conducting an annealing temperature gradient PCR to determine the optimal annealing temperature.
- Reduce the amount of template in the reaction. For high quality DNA, use 1–100 ng genomic DNA or ≤5 ng plasmid/lambda DNA per 50 µL reaction.
- Reduce the number of cycles.
- Reduce the amount of enzyme per reaction.
- Reduce the primer concentration, but not lower than 100 nM of each primer.
- Include DMSO in the reaction to a final concentration of 5%–10%.
1 Koontz, L. Agarose Gel Electrophoresis. Laboratory methods in enzymology : DNA. First edition. edn, Vol. 529 35-45 (2013).
2 Lorenz, T. C. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp, e3998, doi:10.3791/3998 (2012).