Top Questions

My primers are not amplifying the target region after CRISPR genome editing. What could cause this?

We have observed reduced amplification if there is either too much or not enough input genomic DNA (gDNA). Since the concentration of gDNA in samples can vary by cell density and cell type, try repeating your PCR with higher and lower amounts of input gDNA.

In addition, the endogenous DNA repair mechanisms in cells repair double-stranded breaks made by Cas9 in ways that are difficult to predict. It is possible that such repair has altered DNA bases in the primer binding site for your editing detection assay. Try redesigning your primers so that they are further away from the Cas9 cleavage site. Use the design tool at www.idtdna.com/PrimerQuest to help with your design.

Tags:
  • Cas9
  • CRISPR
  • Alt-R
Application Support Topics