Troubleshooting guide for end-point PCR

Sometimes PCR protocol needs optimization to achieve the best results. Please see our PCR troubleshooting guide for suggestions and help with your specific problems. If you have further questions, please contact Solis BioDyne Technical Support:

Problem Possible solution(s)
No or low PCR yield
  • Poor quality of template – check template’s purity and integrity, ensure that your template doesn’t contain PCR inhibitors
  • Template concentration is too low – increase concentration of template
  • Primer concentration is not optimal - titrate primer concentration (final concentration 0.1-0.3 uM of each); ensure that both primers have the same concentration
  • MgCl2 concentration is too low – increase concentration in 0.25 mM increments with a starting concentration of 1.5 mM
  • Primers are degraded – check quality and age of the primers
  • Perform a positive control to ensure that the enzyme, dNTPs and buffers are not degraded and/or contaminated
Non-specific products
  • Use heat-activated, Hot Start polymerase
  • Non-specific amplification - ensure that your primers are target-specific
  • Primer annealing temperature too low - increase annealing temperature; keep your primer annealing temperature 2C to 5C below the Tm of the primer having the lowest Tm
  • Too many cycles - reduce the cycle number by 3-5 to remove non-specific bands
Smearing in electrophoresis
  • Too much template - load lower amount or prepare serial dilutions of template
  • Too many cycles - reduce the cycle number by 3-5
  • Enzyme concentration is too high - decrease the amount of enzyme in final solution in 0.005 U/ul increments (optimal polymerase concentration in final PCR solution is 0.02-0.05 U/ul)
  • Extension time is too long - reduce extension time; Golden Rule for regular DNA polymerases is 1 min/1 kb