Nonspecific Bands or Primer-Dimers (PCR (Polymerase Chain Reaction))

Question

Accepted Answer

Multiple bands appear on gel in addition to or instead of the expected target band. Primer-dimer artifacts or nonspecific amplification products are visible, indicating lack of reaction specificity. Common causes: 1) Too many cycles used (excessive cycling increases nonspecific amplification and errors) 2) Extension or annealing time too long (allows nonspecific amplification and spurious priming) 3) Annealing temperature too low (primers bind nonspecifically; should be 5°C below Tm) 4) Thermal cycler ramping speed too slow (allows spurious annealing during temperature transitions) 5) Primer concentration too high (increases nonspecific binding to template or primer self-binding) 6) Mg2+ concentration too high (increases nonspecific primer binding and unwanted product formation) 7) Calculated primer Tm inaccurate due to incorrect primer concentration calculation Solutions: 1) Use 20–35 cycles; use fewer cycles when template concentration is high 2) Use extension time of 1 min/kb; use annealing time of 30 sec (not longer) 3) Calculate primer Tm using oligocalc with default salt concentration and 0.2–1 µM primer; set annealing temperature 5°C below lowest primer Tm; optimize using thermal gradient 4) Increase thermal cycler to maximum ramp rate if not already set 5) Use well-designed primers at 0.2–1 µM in final reaction; verify correct concentration supplied by manufacturer 6) Reduce amount of Mg2+ in final reaction to minimize nonspecific binding 7) Recalculate primer Tm with correct primer concentration using oligocalc; use lowest Tm for annealing temperature