Artifact Band

Artifact or Nonspecific Bands:

Artifact or non-specific bands are bands that do not correlate to the expected mutant, transgene, or wild type bands. They are the results of primers annealing non-specifically. The presence of such bands can be disconcerting. However, as long as you can still reliable score your results you do not need to be concerned about the presence of the artifacts.

If the presence of artifact bands does impact your ability to accurately score your results as the artifact is too close in size to an expected band or by making the other bands weak or absent, then you will need to address the situation. Some solutions to eliminate or reduce the artifact bands are….

Solution	Rationale
Increase annealing temperature	Increases specificity for primer annealing
Test an annealing temperature gradient	Find the optimal anneal temperature that amplifies expected bands but not artifacts
Decrease primer concentration	Excess primer can lead to nonspecific amplification.
Use a Hotstart Taq polymerase	If primers anneal non-specifically at low temperature as cycle is ramping up, Taq can extend. Inactivating Taq until temperature is high can limit non-specific amplification
Use Touchdown PCR	Starting with a high annealing temperature allows specific bands to amplify initially.
Use a PCR Additive	Reduce DNA secondary structure or non-specific priming