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