Recognition & cleavage sequence

Source: Bacillus stearothermophilus B61

Isoschizomer : Esp3 I

Reaction conditions

1X EzBuffer III, 55℃

1X FastCut Buffer, 55℃

FastCut Buffer

Enzynomics restriction enzyme can cut substrate DNA in 5-15 min with FastCut Buffer

1X EzBuffer III

50 mM Tris-HCl (pH 7.9 @ 25℃), 100 mM NaCl, 10 mM MgCl₂, 100 μg/ml BSA

Unit definition

One unit is defined as the amount of enzyme required for complete digestion of 1-μg bacteriophage λ at 55℃ for 1 hr in 50-μl reaction mixtures.

Storage

10 mM Tris-HCl (pH 7.4 @ 25℃), 300 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 500 μg/ml BSA, 50% glycerol.

Dilution buffer: EzDiluent B

10 mM Tris-HCl (pH 7.4 @ 25℃), 300 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 500 μg/ml BSA, 50% glycerol.

Heat Inactivation

BsmB I can be inactivated at 80℃ for 20 min.

Methylation sensitivity

dam methylation: Not sensitive

dcm methylation: Not sensitive

CpG methylation: sensitive

Prolonged incubation

A minimum amount of enzyme required to digest 1-μg substrate DNA for 16 hr; 0.5 U.

Relative activity in EzBuffers

EzBuffer I: 10%

EzBuffer II: 50%

EzBuffer III: 100%

EzBuffer IV: 25%

FastCut Buffer: 100%

Note

It is an isoschizomer of Esp3 I. Cleavage of mammalian genomic DNA is blocked by CpG methylation.

●  Enzynomics_R075_BsmB I_manual

● Material Safety Data Sheet

- Enzynomics_MSDS_R075_BsmB I

● Quality control

Overdigestion assay

DNA digested for 16 hr in 50-μl reaction with 100 U of enzyme resulted in same the DNA band patterns as those obtained with 1 U of enzyme for 1 hr.

Endonuclease assay

Less than 10% of 1-μg of ΦX174 RFI is converted to RFII when the DNA is incubated with 10 U of enzyme at 37℃ for 4hr in 50-μl reaction. 

Ligation and recutting

More than 95% of DNA fragments (1 μg) digested with 10-fold excess enzyme can be ligated by T4 DNA ligase (400 U) at 16℃ for 16 hr. Of the ligation products, > 95% can be re-cut. 

Extreme pure (EP)

No detectable degradation of ³²P-end labeled single-stranded and double-stranded (5’-, 3’-overhang and blunt end) oligonucleotides occurred during incubation with 100 U of enzyme for 4 hr.