10 mM KH2PO4 (pH 7.45); 200 mM NaCl; 0,1 mM EDTA; 200 µg/ml BSA; 7 mM 2-mercaptoethanol; 50% glycerol.
1X SEBuffer Y
Incubate at 37°C.
1X SEBuffer Y (pH 7.9 @ 25ºC)
33 мМ Tris-Ac, 10 mM MgAc, 66 mM KAc, 1 mM DTT
The enzyme activity depends on number and position of methylated cytosines.
Optimal recognition site:
One unit is defined as the amount of enzyme required to hydrolyze completely a unique site 5`-G(5mC)NG(5mC)-3`/3`-(5mC)GN(5mC)G-5` in 1 μg of
linearized plasmid pFsp4HI3/DriI in 1 hour at 37°C in a total reaction volume of 50μl.
DNA pFsp4HI3/DriI is a linearized plasmid pFsp4HI3, which carries a gene of DNA-methyltransferase M.Fsp4HI and includes a unique GluI site:
Quality Control Assays
No detectable degradation of 1μg of Lambda DNA was observed after incubation with 1 units 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 75-100%
SEBuffer G 75-100%
SEBuffer O 25- 50%
SEBuffer W 50- 75%
SEBuffer Y 100%
SEBuffer ROSE 100%
When using a buffer other than the optimal (suppied) SEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
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