Primers Stop Working Over Time (End-point PCR Primers)

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Accepted Answer

Primers that initially worked successfully for PCR gradually fail or produce poor results after storage Common causes: 1) Repeated freeze-thaw cycles degrading primers 2) Primers forming secondary structures or self-annealing during storage 3) Contamination introduced during repeated opening of stock tubes 4) Degradation due to improper storage conditions Solutions: 1) Aliquot primers for single use before PCR setup 2) Heat aliquoted primers to 94°C for 1 minute before use 3) Quick chill primers on ice after heating before adding to PCR reaction 4) Store primer stocks at -20°C and avoid repeated freeze-thaw cycles 5) Prepare working dilutions and keep frozen stocks unopened