Primer Dimer Formation (PCR / RT-PCR Amplification Problems)

Question

Accepted Answer

Small molecular weight band (typically 50-100 bp) visible at bottom of gel, representing primer self-amplification artifacts that compete with target amplification. Common causes: 1) Excessive primer concentration in reaction 2) Primers possess complementary overlapping sequences enabling self-annealing 3) Annealing temperature too low allowing primer-primer hybridization Solutions: 1) Titrate primer concentration to optimal level for your system 2) Redesign primers to avoid self-complementary internal sequences 3) Increase annealing temperature 4) Use illustra Hot Start Mix PCR products to prevent primer extension during reaction setup