Low PCR Product Yield (End-point PCR Primers)

Question

Accepted Answer

Desired PCR fragment appears faint or barely visible on gel electrophoresis Common causes: 1) Insufficient enzyme amount 2) Denaturation/extension temperature too high causing enzyme degradation 3) Excessive DMSO (>10%) 4) Incorrect annealing temperature 5) Too few PCR cycles 6) Suboptimal Mg2+ concentration 7) Poorly designed primers with mismatched Tm or 3′ complementarity 8) Carryover inhibitors from sample (blood, serum) 9) Insufficient denaturation time for genomic/viral DNA 10) Extension time too short for product size 11) Super-irradiated mineral oil (>2500 mJ/cm2) 12) GC-rich template regions 13) Presence of Taq inhibitors (indigo dyes, heme, melanin) 14) Use of wooden toothpick for colony picking Solutions: 1) Avoid using wooden toothpicks for colony picking or DNA transfer 2) Increase enzyme amount 3) Reduce denaturation/extension temperature 4) Limit DMSO to ≤10% 5) Run temperature gradient starting 5°C below calculated Tm 6) Increase number of cycles 7) Optimize Mg2+ concentration 8) Redesign primers ensuring similar Tm and no 3′ complementarity 9) Remove or dilute inhibitors from sample 10) Extend denaturation time to 10 minutes for genomic/viral DNA 11) Increase extension time appropriate for product size 12) Use mineral oil treated with ≤2500 mJ/cm2 13) For GC-rich templates: eliminate potassium or add 1.0 M betaine with 6–8% DMSO 14) Add BSA (160–600 μg/mL) to counteract Taq inhibitors