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  • FAQ: I don't see any cleavage after my restriction digest. What factors can interfere with cleavage?

    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. DNA methylation is also an important element of a restriction digest.
    If you are having difficulty cleaving your DNA substrate, we recommend the following control reactions. Incubate experimental DNA 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 or adenovirus-2 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.