No Band Due to Incorrect Component Concentrations (PCR (Polymerase Chain Reaction))

Question

Accepted Answer

No visible band or very faint band on gel despite proper thermal cycling parameters. The reaction components may be at suboptimal or inhibitory concentrations. Common causes: 1) dNTP concentration incorrect (too high causes Mg2+ depletion; too low causes insufficient nucleotides) 2) Mg2+ concentration insufficient or omitted (1.5 mM required in final reaction) 3) Primer concentration incorrect (too high increases nonspecific binding; too low causes inefficient annealing; use 0.2–1 µM) 4) Enzyme concentration too low (incomplete replication) or enzyme omitted/inactive 5) Template amount insufficient or template omitted Solutions: 1) Each dNTP should be present at 200 µM in final reaction; verify concentration to avoid Mg2+ depletion 2) Use 1.5 mM Mg2+ in final reaction; ensure component is not omitted 3) Use well-designed primers at 0.2–1 µM in final reaction; verify correct concentration supplied by manufacturer; do not use less than 0.02 µM 4) Use adequate units of enzyme matching template length/difficulty; if enzyme suspected inactive, run PCR with fresh polymerase from different batch 5) Increase template amount or increase cycles in increments of 5; add template if omitted; verify template is present and undamaged