Restriction Endonuclease AarI

For CpG-methylated DNA, cleavage may be inhibited; A 3-hour incubation did not show asterisk activity, but longer digestion may produce asterisk activity; AarI is a complete isoschizase of PaqCI; Complete AarI digestion requires two or more recognition sites; The enzyme volume added to the reaction system should not exceed 10% of the total volume to avoid excessive glycerol in the enzyme causing asterisk activity; Additives (e.g., glycerol, salt) in the restriction endonuclease storage buffer are the same as contaminants (e.g., salt, EDTA, or ethanol) in the substrate solution; the smaller the reaction volume, the stronger the inhibitory effect of the digestion reaction; Isoschizases may have different sensitivities to different methylation modifications; Inactivation conditions: Incubation at 80°C for 20 min. AarI is a Type IIS restriction enzyme and a complete isoschizomer of PaqCI. It recognizes the sequence CACCTGC and cleaves at its downstream site, generating a 5 ' overhang of 4 bases. Unlike conventional Type IIS restriction enzymes, AarI requires two or more recognition sites for efficient digestion. Complete digestion can be achieved with the supplied activator. While digestion can occur in the absence of the activator, full cleavage will not be attained. With a 7-base recognition sequence that occurs infrequently in genes, AarI is particularly suitable for Golden Gate assembly. Concentration: 5 U/μLIsoschizomers: PaqCIRecognition site: CACCTGC (4/8)5 '…CACCTGC(N)?↓…3 '3 '…GTGGACG(N)?↑…5 'Definition of enzyme activity:One unit (U) is defined as the amount of enzyme required to completely digest 1 μg of λDNA in a 50 μL reaction system at 37 °C for 1 hour in the presence of the activator.