10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0,1 mM EDTA; 200 µg/ml BSA;
7 mM 2-mercaptoethanol; 50% glycerol.
SEBuffer W. Incubate at 30°C.
1X SEBuffer W (pH 8.5 @
10 mM Tris-HCl 100 mM NaCl
10 mM MgCl2 1 mM DTT
BlsI cleaves DNA sequence 5’- PuPyNPuPy-3’/3'-PyPuNPyPu-5', if at least two 5-methylcytosines
are present in the recognition site (N isn’t considering).
The enzyme activity varies for different sites and depends on number and
position of methylated cytosines in the recognition sequence.
BlsI doesn’t cleave DNA sequence 5'-A(5mC)NGT-3'/3'-TGN(5mC)A-5'.
Optimal recognition site:
One unit is defined as the amount of enzyme required to hydrolyze at least one of three
5`-G(5mC)NG(5mC)-3`/3`-CGN(5mC)G-5` in 1 μg of linearized
plasmid pFsp4HI3 in 1 hour at 30°C in a total reaction volume of 50
Concentrated enzymes are diluted to approximately 1000 units/ml with the buffer
[10 mM Tris-HCl (pH 7.6); 50 mM KCl; 0.1 mM EDTA; 1 mM DTT; 200 µg/ml BSA; 50% glycerol] before the
is a linearized plasmid pFsp4HI3, which carries a gene
of DNA-methyltransferase M.Fsp4HI and includes three canonical