| 5'-G A C N N^N N G T C-3' |
|
| 3'-C T G N N^N N C A G-5' |
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| Catalog # | B2620 |
| Concentration | 10u/ul |
| Source | Brevibacterium oxydans Iti 12–052 |
| Buffer | 33mM Tris-acetate (pH 7.9), 10mM magnesium acetate, 66mM potassium acetate and 0.1mg/ml BSA. Incubate at 37°C. (Incubation at 30°C results in a 2.5-fold increase in activity.) |
| 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. |
| Storage Buffer | 10mM potassium phosphate (pH 7.4 at 25°C), 100mM NaCl, 1mM EDTA, 7mM 2-mercaptoethanol, 0.2mg/ml BSA and 50% glycerol. |
| Overdigestion Assay | No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with BoxI. |
|
| Ligation/Recutting Assay | After 50-fold overdigestion (3u/ug DNA x 17 hours) with BoxI, more than 90% of the DNA fragments can be ligated at a 5'-termini concentration of 0.08uM. More than 95% of these can be recut. |
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| Labeled Oligonucleotide (LO) Assay | No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10units of restriction endonuclease for 4 hours. |
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| Blue/White Cloning Assay The mix of pUC57/PstI, pUC57/Eco32I and pUC57/HindIII digests was incubated with 10units of enzyme for 5 hours. After religation and transformation 0.2% of white colonies were detected. |
|
| Methylation Effects | The hemi-methylated sequence GAm5C(N)4GTC is cleaved by BoxI at a significantly slower rate. Cleavage is blocked when both strands are methylated as shown: 5'-GAm5C GNNNNm5C GTC-3' 3'-CT Gm5CNNNN Gm5CAG-5' Overlapping CG methylation may influence DNA cleavage. |
|
| Stability during Prolonged Incubation | A minimum of 0.5units of enzyme is required for complete digestion of 1ug of DNA in 16 hours at 37°C. |
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| Thermal Inactivation | Enzyme is inactivated by incubation at 80°C for 20min. |
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| Digestion of Agarose-embedded DNA | A minimum of 10units of enzyme is required for digestion of 1ug of agarose-embedded lambda DNA in 16 hours. |
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| Number of Recognition Sites in DNA | |
|
| Lambda | 7 |
| PhiX174 | 1 |
| M13mp18/19 | 0 |
| pBR322 | 1 |
| pUC18/19 | 0 |
| pUC57 | 0 |
| pTZ19R/U | 0 |
| pBluescriptIIKS(-/+) | 0 |
| pBluescriptIISK(-/+) | 0 |
| pACYC177 | 0 |
| pACYC184 | 2 |
|
| Unit Definition | One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer. |
