Thermo Scientific™ BplI
Cut at ^(8/13)GAG(N)5CTC(13/8)^ sites with BplI restriction enzyme, which performs best at 37°C in Tango(+SAM) buffer.
Manufacturer: Thermo Scientific™ ER1311
Conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.
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
- 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
- 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
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.