Primer Dimer Formation at Low Template Concentrations (PCR (Sigma Guide))

Question

Accepted Answer

Low concentration data points do not fit linear standard curve profile; NTC shows amplification with lower Tm and broader melt peak than positive samples; primer dimers visible on gel, inversely proportional to template concentration Common causes: 1) Primers are self-dimerizing in absence or at low levels of template 2) Excessive primer concentration in reaction 3) Mg2+ concentration too high 4) Annealing temperature too low allowing non-specific primer interactions Solutions: 1) Try lower primer concentrations (test 100 nM, 500 nM, 900 nM) 2) Optimize or re-design the assay to eliminate primer-dimer formation 3) Titrate Mg2+ to determine optimum concentration 4) Perform annealing temperature gradient (55°C to 70°C) to select optimal Ta 5) With RT-PCR, use less RT enzyme, adopt 2-step protocol, or use higher incubation temperature for RT step