AAT Bioquest

How do I increase the intensity of PCR bands?

Posted November 24, 2020


If you are experiencing faint or no bands, try the following tips to increase band intensity:

  • Use the appropriate number of PCR cycles, usually the number of cycles is carried out 25-35 times, but in cases where the template concentration is low use more cycles
  • Optimize the extension time for successful replication of the target, for most PCR applications an extension time of 1 min/kb is sufficient
  • Make sure there is sufficient annealing time for primers to bind to the template
  • Optimize annealing temperature, primers cannot bind to the template if the temperature is too high
  • Use a denaturation temperature of 95 °C
  • Optimize the denaturation time, if the denaturation time is too long or too short, then the DNA might degrade or not completely denature, respectively
  • Each dNTP should have a concentration of 200 µM
  • Optimize your primer concentration, the concentration of each primer should range between 0.1 to 0.5 µM, but for most PCR applications 0.2 µM is sufficient
  • Avoid cross-contamination, contaminated dNTPS and impure water, always use fresh nuclease-free water
  • Make sure to use sufficient amounts of magnesium, for PCR a typical final concentration of magnesium ranges from 1 to 4 mM
Additional resources

Enhancing the specificity of polymerase chain reaction by graphene oxide through surface modification: zwitterionic polymer is superior to other polymers with different charges

Polymerase Chain Reaction (PCR)

Gelite™ Orange Nucleic Acid Gel Staining Kit