Restriction digestion, also known as restriction endonucleases, is the process of cutting DNA at a specific site, determined by the surrounding DNA sequence. Restriction digestion is accomplished by warming the target DNA molecule with a restriction enzyme (an enzyme that recognizes and binds to a specific DNA sequence and cuts a specific nucleotide within or outside the recognition sequence). Restriction digestion can result in the production of flat ends (DNA molecule ends ending in base pairs) or sticky ends (DNA molecule ends ending in nucleotide protrusions). Components of a typical restriction digestion reaction include a DNA template, a selected restriction endonuclease, a buffer, and sometimes a BSA protein. The reaction is incubated at the specific temperature required for optimal restriction enzyme activity and terminated by heating.Creative Enzymes provides you with a variety of Troubleshooting Restriction Enzyme Digestions to meet your scientific research needs.
| Probable Cause | Solutions |
| Dirty template DNA | We use a specific cleanup system to clean up the substrate DNA. alternatively, phenol/chloroform extraction followed by ethanol precipitation can be used to purify the DNA substrate. |
| Presence of inhibitors | We use specific methods to remove enzyme inhibitors from substrate DNA solutions (e.g., SDS, phenol, EDTA, chloroform, ethanol, CsCl, high salt, or plasticizers in microcentrifuge tubes). Alternatively, phenol/chloroform extraction and/or ethanol precipitation can be used to remove inhibitors from DNA preparations. |
| DNA methylated | We provide information on the sensitivity of restriction enzymes to the methylation status of substrates. For plasmid DNA, methylation is eliminated by passing the DNA to a bacterial host that lacks the interfering methylesterase. |
| DNA unmethylated | Some enzymes require methylation of their target sites. For example, Dpn I requires N6-methylation of adenine residues to function. |
| Incorrect sequence information | Double check sequence information to confirm the number and location of enzyme recognition sites. |
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