Description: EcoRI Methyltransferase modifies the internal adenine residue (N6) of the sequence GAATTC. Source: An E. coli. strain that carries the cloned EcoRI modification gene from Escherichia coli RY13 (R. N. Yoshimori) Reagents Supplied: EcoRI Methyltransferase Reaction Buffer (10X) S-adenosylmethionine (SAM) Molecular Weight: Theoretical: 37,911 daltons
Reaction Conditions: 1X EcoRI Methyltransferase Reaction Buffer Supplemented with 80 μM S-adenosylmethionine (SAM) Incubate at 37°C.
1X EcoRI Methyltransferase Reaction Buffer: 50 mM Tris-HCl
50 mM NaCl
10 mM EDTA
pH 8.0 @ 25°C
Unit Definition: One unit is defined as the amount of enzyme required to protect 1 μg λ DNA in 1 hour at 37°C in a total reaction volume of 10 μl against cleavage by EcoRI restriction endonuclease.
Protection Assay Conditions: EcoRI Methyltransferase is incubated with 1 µg of λ DNA in 10 µl 1X EcoRI Methyltransferase Buffer, supplemented with 80 µM S-adenosylmethionine, for one hour at 37°C followed by 15 minutes at 65°C. The extent of protection by EcoRI Methyltransferase is determined by the addition of 40 µl NEBuffer 2 and 5 units of EcoRI restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on an agarose gel.
Concentration: 40,000 units/ml
Storage Conditions: 100 mM KPO4
200 mM NaCl
10 mM 2-Mercaptoethanol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C