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You are here:Home » Molecular Biology » MB-Enzymes, Restriction » BcuI (SpeI)

BcuI (SpeI)


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5'-A^C T A G T-3'
3'-T G A T C^A-5'
Catalog #B0808-05
Concentration 10u/ul
Source Bacillus coagulans VS 29-022
Unit Definition One unit is defined as the amount of B0808-05 required to digest 1ug of control DNA fragments in 1 hour at 37°C in 50ul of assay buffer. The control DNA is pUC19 DNA with inserted BcuI recognition site - Psp1406I fragments.
Storage Buffer Supplied as a liquid in 10mM Tris-HCl (pH 7.5 at 25°C), 300mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
Supplied withR1625: Restriction Enzyme Buffer A, 10X: Suplied as a liquid in 33mM Tris-acetate pH 7.9, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA. Incubate at 37°C.
Diluent Buffer 10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol. For longer periods–the Storage Buffer should be used.
Overdigestion AssayNo detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug pUC19-BcuI DNA x 16 hours) with BcuI.
Ligation/Recutting (L/R) AssayThe ligation and recleavage assay was replaced with LO test after validating experiments showed LO test ability to trace nuclease and phosphatase activities with sensitivity that is higher than L/R by a factor of 100.
Labeled Oligonucleotide (LO) AssayNo detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of BcuI for 4 hours.
Blue/White (B/W) Cloning AssayThe B/W assay was replaced with LO test after validating experiments showed LO test ability to detect nuclease and phosphatase activities with sensitivity that equals to that of B/W test.
Stability during Prolonged IncubationA minimum of 0.5units of BcuI is required for complete digestion of 1ug of Ad2 DNA in 16 hours at 37°C.
Methylation Effects on Digestion
Dam, Dcm, CpG - Never overlaps, no effect
EcoKI, EcoBI - effect not determined
Digestion of Agarose-embedded DNAA minimum of 10 units of BcuI is required for complete digestion of 1ug of agarose-embedded Adenovirus-2 DNA in 16 hours.
Compatible EndsEco130I, NheI, XbaI, XmaJI
Number of Recognition Sites in DNA
Thermal InactivationBcuI is not inactivated by incubation at 80°C for 20min.
Inactivation Procedure
1. To prepare the digested DNA for electrophoresis(a) stop the digestion by adding 0.5M EDTA, pH 8.0, to achieve a 20mM final concentration. Mix thoroughly, add an electrophoresis loading dye and load onto gel.
2. To prepare DNA suitable for further enzymatic reactions(a) extract with phenol/chloroform, precipitate with ethanol or isopropanol, wash the pellet with 75% cold ethanol and air dry; (b) dissolve DNA in either nuclease-free water, TE buffer, or a buffer suitable for further applications; (c) check the DNA concentration in the solution.

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