FAQ: PCR Tips & Tricks

Tip & Tricks Little or No PCR Amplification Multiple/Smeared PCR Amplification
Annealing Temperature Lower annealing temperature by 2°C Increase annealing temperature by 2°C
Hot Start Prepare reactions on ice; place reactions directly on preheated thermo cycler Prepare reactions on ice; place reactions directly on preheated thermo cycler
Touchdown/Stepdown PCR Perform initial cycles above calc. TM; lower annealing temp 1-4°C, every other cycle to ~10°C below calc. TM Perform initial cycles above calc. TM; lower annealing temp 1-4°C, every other cycle to ~10°C below calc. TM
Primer Design Design primers with higher annealing temperatures without hairpin loops or primer dimers Design primers with higher annealing temperatures without hairpin loops or primer dimers
Primer Degradation Check by electrophoresis on denaturing acrylamide gel Check by electrophoresis on denaturing acrylamide gel
Cycle Conditions Increase number of cycles by 5 Decrease number of cycles by 5
Denaturing Time Increase up to 98°C. Increase initial denaturation by 5 min or denature 72°C for 10 min in 50mM NaOH  
PreMix Concentrations
  Use up to 104-106 molecules/reaction (nanograms for cloned templates; micrograms for genomic DNA). Decrease enzyme and/or primer volumes