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)

Quality Control Assays

16-Hour Incubation:

No detectable degradation of 1μg of Lambda DNA was observed after incubation with 1units of enzyme for 16 hours at 37°C in a total reaction volume of 50 μl.

Oligonucleotide Assay:

No detectable degradation of a single- and double-stranded oligonucleotide was observed after incubation with 1 units of enzyme for 3 hours.

Enzyme Properties

Activity in SEBuffers:

SEBuffer B       50-75%

SEBuffer G        100%

SEBuffer O      25-50%

SEBuffer W      50-75%

SEBuffer Y      75-100%

SEBuffer ROSE  100%

When using a buffer other than the optimal (suppied) SEBuffer, it may be necessary to add more enzyme to achieve a complete digestion.