Incorrect PCR Product Size (PCR (Polymerase Chain Reaction))

Question

Accepted Answer

Gel electrophoresis shows PCR product band(s) at unexpected molecular weight, either larger or smaller than the predicted amplicon size. Common causes: 1) Incorrect annealing temperature causing non-specific primer binding 2) Mispriming due to primers having complementary regions within template DNA 3) Suboptimal Mg²⁺ concentration affecting polymerase processivity and specificity Solutions: 1) Recalculate primer Tm values using NEB Tm calculator and adjust annealing temperature accordingly 2) Verify primers have no additional complementary regions within template DNA using BLAST or primer design software 3) Optimize Mg²⁺ concentration by testing increments of 0.2–1 mM 4) Repeat reactions using fresh reagent solutions to eliminate nuclease contamination