Resources » Technical Resource Centers » Gene Technical Resources » Molecular Cloning Center » Restriction Digestion Troubleshooting Guide
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 |
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Enzyme quality is compromised |
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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.
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Incomplete or no template/vector digestion |
Not enough incubation time |
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Template/plasmid contamination with inhibitors |
Inhibitor contamination can originate from miniprep kits, or leftover PCR reaction components:
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Enzyme concentration too low |
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Incompatible buffer was used |
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Digestion product is smeared on gel |
Template is contaminated with nucleases |
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Enzyme is bound to DNA substrate |
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Template/vector appears to be over digested |
Star activity (enzyme cleaved similar recognition sequence) |
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