Nonspecific Amplification and Smearing on Gel (PCR (Invitrogen Guide))

Question

Accepted Answer

Gel shows multiple bands, smears, or high background instead of single clean product band. May include primer-dimers at bottom of gel. Common causes: 1) Excess DNA template input or degraded DNA creating smears 2) Primers containing complementary sequences or consecutive G/C at 3′ ends causing primer-dimers 3) High primer concentration (>1 μM) promoting primer-dimer formation 4) Excess DNA polymerase or Mg2+ concentration favoring nonspecific binding 5) Annealing temperature too low (<3–5°C below Tm) or annealing time too long 6) Too many PCR cycles accumulating nonspecific products Solutions: 1) Lower DNA template quantity; evaluate integrity by gel electrophoresis and store in TE buffer (pH 8.0) 2) Redesign primers avoiding complementary sequences and consecutive G/C at 3′ ends; consider nested PCR or longer primers 3) Optimize primer concentrations to 0.1–1 μM range 4) Reduce DNA polymerase and optimize Mg2+ concentration for each primer set 5) Increase annealing temperature to 3–5°C below lowest primer Tm; shorten annealing time; consider touchdown PCR 6) Use hot-start DNA polymerases or set up on ice; reduce cycle number without drastically lowering yield