DNA Methyltransferase M.BseCI
(concentration 5u/μl)

5'…ATCGAT…3'

Description: M.ΒseCI modifies the N6 atom of the 3΄ adenine residue in the sequence 5΄-ATCGAT-3΄.

Source: M.BseCI is a methyltransferase purified from an E. coli strain that carries the BseCI methyltransferase gene (bseCIM) from Bacillus stearothermophilus, cloned in plasmid pBseCIM8 (1,2).

Reaction Buffer: 10 mM Tris-HCl (pH 7.4), 10 mM EDTA, 5 mM 2-mercaptoethanol, 0.02% Triton-X-100.

Unit definition: One unit is defined as the amount of enzyme required to protect 1μg of λ DNA in 1 hour at 55^oC in a total reaction volume of 10μl against cleavage by BseCI restriction endonuclease.

Protection Assay Conditions: M.BseCI is incubated with 1μg of λ DNA in 10μl 1x M.BseCI buffer, supplemented with 80μM S-adenosylmethionine (SAM), for 1 hour at 55^oC followed by 15 minutes at 70^oC. The extent of protection by M.BseCI is determined by the addition of 40μl BseCI Reaction Buffer and 10 units of BseCI restriction endonuclease. Incubation at 55^oC for 30 minutes is followed by analysis on agarose gel.

Note: M.BseCI exhibits 35% activity at 37^oC.

Storage buffer: 50 mM Tris-HCl (pH 7.4), 10 mM EDTA, 1 mM dithiothreitol, 200 μg/ml BSA and 50% glycerol. Store at -20^oC.

Quality Control: Tested for the absence of endo- and exodeoxyribonucleases.

References: 1. Rina, M. and Bouriotis, V. (1993) Cloning, purification and characterization of the BseCI DNA methyltransferase from Bacillus stearothermophilus. Gene 133, 91-94.

2. Rina, M., Markaki, M. and Bouriotis, V. (1994) Sequence of the cloned bseCIM gene: M.BseCI reveals high homology to M.BanIII Gene 150, 71-73.

Reagents supplied: 10x M.BseCI buffer