DNA digestion by Restriction Enzymes  can be a sensitive process dependent on the concentrations of the reactions components and reaction time. Use the troubleshooting guide below to optimize your restriction digestion reactions  or get your desired gene in the vector you want the easy way with GenEZ™ ORF Clones. Start with a Search for your gene.

Problem Causes Solutions
 
Enzyme quality is compromised
  • Check enzyme expiration date.
  • Avoid multiple freeze thaw cycles of enzyme.
Methylation of template sequence prevents enzyme from cutting
DNA from a bacterial source or eukaryotic source may be blocked by Dam and Dcm methylation or CpG methylation, respectively.
  • Use a Dam- or Dcm- strain.
Incomplete or no template/vector digestion
Not enough incubation time
  • Add 1 to 2 hours to incubation time.
Template/plasmid contamination with inhibitors
Inhibitor contamination can originate from miniprep kits, or leftover PCR reaction components:
  • Use dialysis or spin column to remove contamination.
  • Dilute or use less volume of DNA - DNA solution should compose no more than 25% of digestion reaction.
  • Use PCR clean up kit to remove PCR reaction components.
Enzyme concentration too low
  • Use 3–5 units of enzyme / μg of DNA.
Incompatible buffer was used
  • Use recommended buffer with enzyme.
Digestion product is smeared on gel
Template is contaminated with nucleases
  • Use commercial kit to clean up template.
  • Use running buffer and agarose gel made with fresh nuclease-free water.
Enzyme is bound to DNA substrate
  • Decrease units of enzyme.
  • Remove enzyme from DNA by adding 0.1–0.5% SDS to loading buffer.
Template/vector appears to be over digested
Star activity (enzyme cleaved similar recognition sequence)
  • Use minimum incubation time.
  • Reduce number of enzyme units in reaction.
  • Use compatible buffer with enzyme.
  • Use a high-fidelity enzyme.

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