No Band Due to Template or Reagent Quality Issues (PCR (Polymerase Chain Reaction))

Question

Accepted Answer

PCR fails to produce visible band despite correct concentrations and cycling parameters. Template degradation, contamination, or impure reagents may be inhibiting the reaction. Common causes: 1) Template damaged, degraded (sheared), or contains PCR inhibitors 2) Primers contain impurities that inhibit PCR 3) Impure dNTPs used (contaminants cause incomplete/incorrect amplification or inhibition) 4) Water contaminated during prior pipetting events 5) PCR product has high GC content (>65%) making amplification difficult Solutions: 1) Use fresh template and increase cycles; if inhibitors suspected, dilute existing template; run control reaction with pure plasmid plus template to determine inhibitory effects 2) Use desalted primers or more highly purified primers; try diluting primers to test for inhibitory effects (but maintain at least 0.02 µM) 3) Use high-quality dNTPs from reliable source 4) Use fresh nuclease-free water 5) For GC-rich templates (>65%), increase annealing temperature and optimize using thermal gradient; add DMSO or secondary structure destabilizer (do not exceed 10%)