Thermo Scientific™ BplI (5 U/μL)

5'...▵ ₈(N) G A G (N)₅ C T C (N)₁₃▵...3'

3'...▵₁₃(N) C T C (N)₅ G A G (N)₈ ▵...5'

Conditions for 100% Activity:

• [1X Buffer Tango™] + SAM:[33mM Tris-acetate (pH 7.9 at 37°C), 10mM Mg-acetate, 66mM K-acetate and 0.1mg/mL BSA] + 0.05mM S-adenosylmethionine
• Incubate at 37°C

Storage Buffer:

• BplI is supplied in:10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/mL BSA and 50% (v/v) glycerol

Ligation and Recleavage:

• After 10-fold overdigestion with BplI, more than 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme

Methylation Effects:

• Dam: never overlaps — no effect
• Dcm: never overlaps — no effect
• CpG: may overlap — no effect
• EcoKI: never overlaps — no effect
• EcoBI: never overlaps — no effect

Digestion of Agarose-embedded DNA:

• Minimum 10units of the enzyme are required for complete digestion of 1μg of agarose-embedded lambda DNA in 16hours

Note:

BplI requires only Mg² + for its activity, but is stimulated by S-adenosylmethionine. 0.05mM S-adenosylmethionine gives more than a 100-fold increase in BplI activity. Still, complete cleavage of some substrates with BplI is difficult to achieve. BplI concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase.