Restriction enzymes were first discovered in bacteria, but were later discovered in some archaea. Typically, restriction enzymes cut double-stranded DNA. Each restriction endonuclease recognizes a specific DNA sequence, and the cut can occur within the recognized sequence or some distance away, depending on the enzyme. Creative Enzymes provides you with a variety of restriction enzyme substrate research services to meet your scientific research needs.
We will investigate restriction enzyme substrates in the following ascepts:
| Substrate source and structure | Substrates typically used for restriction enzyme digestion include phage DNA, plasmid DNA, genomic DNA, PCR products and double-stranded oligonucleotides. the concentration of the DNA sample affects the success of restriction digestion. A viscous DNA solution produced from a large volume of DNA that is too small can inhibit diffusion and significantly reduce enzyme activity |
| Plasmid DNA | Round superhelical plasmid DNA is typically 3-10 kb in size, and plasmids typically require more units of restriction enzyme to fully cleave than linear DNA due to the superhelix or the total number of sites to be digested. Fewer enzymes may be required for digestion if the superhelical plasmid is first linearized with another restriction enzyme or relaxed with a topoisomerase. |
| Genomic DNA | Digestion of genomic DNA can be difficult due to methylation and viscosity. We can adjust the viscosity by increasing the reaction volume. Digestion is usually more efficient when genomic DNA is diluted to a minimum concentration of 10 µg per 50-200 µl. |
| PCR products | PCR-amplified DNA can be digested with restriction enzymes that have recognition sequences in the amplification sequence or primer region. The number of enzyme units required must be balanced against the total number of loci to ensure complete cleavage. Longer incubation times may be required to ensure complete digestion. We should avoid using low overdigestion values in overnight digestions. |
| Recognition site density | Restriction enzyme activity units are usually defined based on a one hour digestion of 1 µg of λ DNA. When digesting other substrates, we adjust for the amount of substrate, the number of recognition sites per molecule, and the incubation time. |
Creative Enzymes has accumulated years of quintessence in the field of enzyme folding analysis service, helping our clients accelerate drug discovery and development and improve the overall success rate of their projects. If you would like to know more about this service, please feel free to contact us.
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