Kro I

Supplied in: 10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0,1 mM EDTA; 200 mg/ml BSA; 7 mM  2-mercaptoethanol; 50% glycerol.

Reaction Conditions: 1XSEBuffer G.

Incubate at 37°C.

1XSEBuffer G (pH 7.6 @ 25ºC)

10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT

Substrate specificity: The enzyme cleaves C5-methylated DNA and doesn’t cut unmodified DNA [1]. Kro I doesn`t cleave DNA modified with MspI DNA methyltransferase.

Unit Definition: One unit is defined as the amount of enzyme required to hydrolyze completely1 μg of linearized plasmid pMHpaII 1 in 1 hour at 37°C in a total reaction volume of 50 μl. DNA pMHpaII 1/DriI is a linearized plasmid pMHpaII 1. pMHpaII 1 carries a gene of DNA-methyltransferase M.HpaII, which methylates sites 5`-CCGG-3` producing 5`-C(5mC)GG-3`/3`-GG(5mC)C-5`, and includes three canonical sites 5`-GC(5mC)GGC-3`/3`-CGG(5mC)CG-5`.

Reagents supplied with enzyme: 10XSEBuffer G

Heat Inactivation: Yes   (65°C for 20 minutes)