Recognition site DNA sequence with at least two 5mC:
Sourse: Bacillus simplex 23
The enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA
10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0,1 mM EDTA; 200 µg/ml BSA; 7 mM 2- mercaptoethanol; 50% glycerol.
1X SEBuffer W.
Incubate at 30°C.
1X SEBuffer W (pH 8.5 @ 25ºC):
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
Optimal recognition site:
One unit is defined as the amount of enzyme required to hydrolyze at least one of three canonical sites:
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 μl. 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 activity determination. DNA
pFsp4HI3/DriI is a linearized plasmid pFsp4HI3, which carries a gene of DNA-methyltransferase M.Fsp4HI and includes three canonical sites: 5`-G(5mC)NG(5mC)-3`/3`-CGN(5mC)G-5`.
Quality Control Assays
No detectable degradation of 1μg of Lambda DNA was observed after incubation with 10 units of enzyme for 16 hours at 30°C in a total reaction
volume of 50 μl.
No detectable degradation of a single- and double-stranded oligonucleotide was observed after incubation with 5 units of enzyme for 3 hours.
Activity in SEBuffers:
SEBuffer B 10-25%
SEBuffer G 10-25%
SEBuffer O 50-75%
SEBuffer W 100%
SEBuffer Y 75-100%
SEBuffer ROSE 50%
When using a buffer other than the optimal (supplied) SEBuffer, it may be necessary to add more enzyme to achieve a complete digestion.
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