General PCR reminders

  • When collecting tail tips, clean tools with 70% ETOH between animals.
  • An assay that works well in one lab may not work well in another. Conditions and components need to be optimized for your lab conditions.
  • Use filtered pipette tips.
  • Avoid repeated freezing and thawing of nucleotides, aliquot small volumes and store at -70°C.
  • Mix the reaction tube by gentle tapping. Do not vortex PCR mix. 
  • Add DNA polymerase (Taq) to the reaction tube last.
  • Adjust electrophoresis voltage and run time to improve band resolution.
  • Confirm that the PCR machine was programmed correctly.
  • If bands are weak, try using a longer extension time.
  • If there are more bands than expected (or non-specific bands), try increasing or decreasing the annealing temperature.
  • Avoid overloading PCR products into the gel; this may result in cross-contamination or misinterpretation of the results.
  • Do NOT perform PCR in a ventilated hood as it increases risk of cross-contamination.