Restriction enzymes.- For complette list, please click here: Overview
Description	Restriction enzymes are endonucleases that recognize specific ds DNA sequences and cleave the DNA in both strands. Three classes are recognized: Type I (EC 3.1.21.3.) cleave the DNA at apparently random sites far away from the recognition site. Type III enzymes (EC 3.1.21.5.) cleave the DNA outside of the recognition site. Most characterized enzymes belong to the Type II class (EC 3.1.21.4); together with the Type IIS class they comprise the commercially available restriction enzymes used for DNA analysis and manipulation cleavage within the recognition site. Type II enzymes (EC 3.1.21.4) recognize symmetric DNA sequences and cleave within the sequences, leaving a 3´-hydroxyl on one side of the cut and a 5´-phosphate on the other. Type IIS enzymes recognize asymmetric and uninterrupted sequences, 4-7 bp in length. They cleave at a defined distance, up to 20 bp, to one side of their recognition sequence.
Unit definition	1U is defined as the activity that cleaves 1 µg of lambda DNA in a 50µl reaction in 1 h under optimum buffer conditions at optimum temperature (usualy 37°C).
Reaction buffers	Each enzyme supplied with a 10x reaction buffer: Reaction buffer A (10X): 660 mM K-acetate, 330 mM Tris-acetate pH 7.9, 100 mM Mg-acetate, 5 mM DTT.  Reaction buffer B (10X): 1 M NaCl, 100 mM Tris-HCl pH 8.0, 50 mM MgCl2, 10 mM 2-mercaptoethanol. Reaction buffer M (10X): 500 mM NaCl, 100 mM Tris-HCl pH 7.5, 100 mM MgCl2, 10 mM DTE. Reaction buffer L (10X): 100 mM Tris-HCl pH 7.5, 100 mM MgCl2, 10 mM DTE. Reaction buffer H (10X): 1 M NaCl, 100 mM Tris-HCl pH 7.5, 100 mM MgCl2, 10 mM DTE.
Reaction conditions	The preparation of DNA to be cleaved should be free of contaminants such as phenol, chlorophorm, alcohol, EDTA, detergents, or excessive salts, all of which can interfere with restriction endonuclease activity. The activity and specificity of some REs are influenced by the methylation state of the DNA substrate. To avoid site-specific dam and/or dcm methylation of recognition sites in E. coli vectors, it is necessary to propagate the vectors in strains lacking one or both of the methyltransferase systems. It must be taken into account, however, that E. coli strains, deficient in dam or dcm methylation, have elevated rates of mutation.   ”Star“ activity is defined as a relaxed specificity of restriction enzymes under suboptimal reaction conditions (e.g. high glycerol concentration, low ionic strength or high pH values in the reaction buffer) and is related to the naturally observed limited accuracy of these enzymes when used in high concentration over a prolonged time. To keep glycerol concentration at less than 5% in a reaction, the restriction endonuclease should not exceed 10% of the total reaction volume. Depending on the specific purpose, the digestion reaction is terminated by inhibiting, destroying or extracting the restriction enzyme. This can be achieved by chelation of the essential cofactor Mg2+, thermal denaturation (usualy, at 65° to 95° for 10 min), or by phenol/chloroform extraction.
Storage	Store at -20°C.
Applications	Applications: analysis of the methylation state of genes (e.g., Hpa II and Msp I); RFLP analysis; genetic fingerprinting; cloning, restriction mapping, DNA labeling.
Quality control	activity, purity, absence of nonspecific endonucleases/exonucleases/other specific restriction endonucleases/phosphatases, ligation-recut assay.
References	1. http://rebase.neb.com