Glu I

Supplied in:

10 mM KH2PO4 (pH 7.45); 200 mM NaCl; 0,1 mM EDTA; 200 µg/ml BSA; 7 mM  2-mercaptoethanol; 50% glycerol.

Reaction Conditions:

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

Substrate specificity:

The enzyme activity depends on number and position of methylated cytosines.

Optimal recognition site:

5'-G(5mC)↑NG(5mC)-3'/3'-(5mC)GN↓(5mC)G-5`

Unit Definition:

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:

5`-G(5mC)NG(5mC)-3`/3`-(5mC)GN(5mC)G-5` [2].

Quality Control Assays

16-Hour Incubation:

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.

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        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.

Reagents supplied with enzyme:

10XSEBuffer Y

Heat Inactivation:

Yes   (80°C for 20 minutes)