No Band Due to Omitted Critical Components (PCR (Polymerase Chain Reaction))

Question

Accepted Answer

Complete absence of PCR product on gel due to missing essential reaction components, representing a critical setup error. Common causes: 1) dNTPs omitted from reaction (no nucleotides for DNA synthesis) 2) Primer(s) omitted from reaction (no initiation of amplification) 3) Template DNA omitted from reaction (no target for amplification) 4) Polymerase enzyme omitted or inactive (no catalytic activity for DNA synthesis) 5) Mg2+ omitted from reaction (cofactor essential for polymerase activity) Solutions: 1) Add each dNTP at 200 µM final concentration 2) Add well-designed primers at 0.2–1 µM final concentration 3) Add appropriate amount of template DNA 4) Add adequate units of active polymerase; test with fresh enzyme from different batch if suspected inactive 5) Add Mg2+ to 1.5 mM final concentration