Nonspecific Bands or Primer-Dimers Due to Thermal Cycle Issues (PCR (Polymerase Chain Reaction))

Question

Accepted Answer

Gel shows multiple bands instead of single target band, or primer-dimers appear as small molecular weight products. The desired product may be present but accompanied by unwanted amplification artifacts. Common causes: 1) Too many cycles used (>35 cycles) increasing opportunity for nonspecific amplification and errors 2) Annealing temperature too low allowing primers to bind nonspecifically to template 3) Annealing time too long (>30 sec) increasing spurious priming events 4) Extension time too long (>1 min/kb) allowing nonspecific amplification 5) Thermal cycler ramping speed too slow allowing spurious annealing during temperature transitions 6) Primer Tm calculated inaccurately leading to incorrect annealing temperature selection Solutions: 1) Use 20–35 cycles; fewer cycles when template concentration is high 2) Set annealing temperature 5°C below lowest primer Tm; optimize using thermal gradient for greater accuracy 3) Use annealing time of 30 sec (not longer) 4) Use extension time of 1 min/kb (not longer unless target requires) 5) Increase thermal cycler ramp rate to maximum speed if not already set 6) Recalculate primer Tm using oligocalc with default salt concentration and actual primer concentration (0.2–1 µM)