There can be several different reasons why your restriction enzyme does not cut the DNA as reviewed in this video.
The preparation of DNA to be cleaved should be free of contaminants such as phenol, chloroform, alcohol, EDTA, detergents, or excessive salts, all of which can interfere with restriction enzyme activity. Monarch Nucleic Acid Purification Kits may be used to purify sample DNA prior to digestion.
If you are having difficulty cleaving your DNA substrate, we recommend the following control reaction:
Incubate experimental DNA in reaction buffer without restriction enzyme (degradation of DNA indicates contamination in the DNA preparation or
reaction buffer) and control DNA (DNA with multiple known sites for the enzyme, e.g. lambda DNA) with restriction enzyme to more accurately judge
whether or not the reaction went to completion. If the control DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample. If an inhibitor (often salt, EDTA or phenol) is present, the control DNA will not cut after mixing. Additional troubleshooting help is also available.
DNA methylation is also an important element of a restriction digest. If methylation inhibition is suspected, please refer to the link describing dam, dcm and CpG methylation.
