Unexpected PCR Product (End-point PCR Primers)

Question

Accepted Answer

PCR produces a band of incorrect size or sequence not matching the expected target Common causes: 1) Poor primer design allowing binding at alternative sites 2) Three or more consecutive G or C nucleotides at 3′ end causing mispriming 3) Annealing temperature too low reducing specificity 4) Excessive Mg2+ concentration 5) DNA contamination from previous reactions or environment Solutions: 1) Redesign primers: use longer primers to avoid alternative binding sites 2) Avoid 3 consecutive G or C nucleotides at 3′ primer end 3) Increase annealing temperature to enhance specificity 4) Lower Mg2+ concentration 5) Prevent contamination: use aerosol-resistant tips and separate work areas 6) Implement UNG/UDG technique to prevent carryover contamination