10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0,1 mM EDTA; 200 mg/ml BSA; 7 mM 2-mercaptoethanol; 50% glycerol.
Incubate at 37°C.
1XSEBuffer G (pH 7.6 @ 25ºC)
10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT
The enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA . Kro I doesn`t cleave DNA modified with MspI DNA methyltransferase.
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:
Yes (65°C for 20 minutes)
Quality Control Assays
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.
No detectable degradation of a single- and double-stranded oligonucleotide was observed after incubation with 1 units of enzyme for 3 hours.
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.