False Positive Amplification from Contamination (PCR (Invitrogen Guide))

Question

Accepted Answer

PCR products appear in negative control reactions without template. Products may appear in samples expected to be negative. Common causes: 1) Primers containing complementary sequences causing self-oligomerization and amplification 2) Crossover contamination of DNA in work environment 3) Carryover contamination from previous PCR products 4) Non-dedicated pipettes or work areas spreading amplicons Solutions: 1) Redesign primers avoiding complementary and self-complementary sequences that favor primer-dimer formation 2) Follow general PCR setup recommendations; dedicate separate work area and decontaminate after each use 3) Use pipette tips with aerosol barriers for all pipetting steps 4) Implement dUTP incorporation with UDG treatment for carryover control 5) Maintain separate pre-PCR and post-PCR areas with dedicated equipment