Traditional molecular cloning
Restriction enzyme overview
- General information about restriction enzymes
- Star activity of restriction enzymes
- Inactivation of restriction enzymes
- Buffer activity with restriction enzymes
- Universal buffers for double digestion with restriction enzymes
- Restriction enzymes affected by methylation
- Methylation-sensitive restriction enzymes
- QC of restriction enzymes
- Ligation cloning overview
- Ligation product guide
General information about restriction enzymes
Our restriction enzymes are classified into three categories, according to their cofactor requirements and the characteristics of their cleavage sites:
|Type||Cofactor(s)||Cleavage site||Example enzymes|
|Type I||ATP, AdoMet, Mg2+||Recognition sites and cleavage sites are different; cleavage sites are not fixed||EcoB, EcoK|
|Type II||Mg2+||These enzymes cleave DNA at the recognition sequence or at a defined distance from the recognition site||EcoRI, BamHI|
|Type III||ATP, Mg2+||These enzymes cleave DNA at fixed sites, although their recognition sites and cleavage sites are different||EcoPI, HinfIII|
Type II restriction enzymes are generally used in genetic engineering experiments. Restriction enzymes induce cleavage and/or star activity, depending on the substrate DNA and reaction conditions. Close attention should be paid to the factors described below.
Under certain reaction conditions, a restriction enzymes can lose its specificity and cleave base sequences that are different from the original recognition sites. This phenomenon is called star activity, and while almost all restriction enzymes have star activity, its occurrence depends on the enzyme, substrate DNA, and reaction conditions. In addition to this relaxation of recognition sites, nicking activity—in which substrate DNA is partially cleaved—is also observed. In order to suppress star activity, we recommend setting up restriction digests at lower glycerol concentrations, neutral pH, and higher salt concentrations. Note, though, that these conditions may also create lower reactivity overall.
For additional information, please refer to our page about compensating for star activity.
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