| 5'-A^C T A G T-3' |
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| 3'-T G A T C^A-5' |
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| 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 with | R1625: 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. |
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| Overdigestion Assay | No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug pUC19-BcuI DNA x 16 hours) with BcuI. |
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| Ligation/Recutting (L/R) Assay | The 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. |
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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 10 units of BcuI for 4 hours. |
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| Blue/White (B/W) Cloning Assay | The 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. |
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| Stability during Prolonged Incubation | A minimum of 0.5units of BcuI is required for complete digestion of 1ug of Ad2 DNA in 16 hours at 37°C. |
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| Methylation Effects on Digestion | |
| Dam, Dcm, CpG - Never overlaps, no effect |
| EcoKI, EcoBI - effect not determined |
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| Digestion of Agarose-embedded DNA | A minimum of 10 units of BcuI is required for complete digestion of 1ug of agarose-embedded Adenovirus-2 DNA in 16 hours. |
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| Compatible Ends | Eco130I, NheI, XbaI, XmaJI |
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| Number of Recognition Sites in DNA | |
| Ad2 | 3 |
| Lambda | 0 |
| PhiX174 | 0 |
| M13mp18/19 | 0 |
| pBR322 | 0 |
| pUC18/19 | 0 |
| pUC57 | 0 |
| pTZ19R/U | 0 |
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| Thermal Inactivation | BcuI is not inactivated by incubation at 80°C for 20min. |
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| 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. |
