| 5'-A T T T^A A A T-3' |
|
| 3'-T A A A^T T T A-5' |
|
| Catalog # | S1014-59 |
| Source | Streptococcus milleri S |
| Concentration | 10units/ul |
| Unit Definition | One unit is defined as the amount of SmiI required to digest 1ug of Ad2 DNA-SspI fragments in 1 hour at 30°C in 50ul of assay buffer. |
| Storage Buffer | 10mM Tris-HCl (pH 7.4, 25°C) 200mM NaCl, 1mM DTT, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol. |
| Supplied With | R1625 Restriction Enzyme Buffer A: 10X: 10mM Tris-HCl (pH 7.4, 25°C) 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol. For longer periods, the Storage Buffer should be used. |
| R1625-03 Restriction Enzyme Buffer D | 10X: 50mM Tris-HCl, pH 7.5, 10mM MgCl2, 100mM sodium chloride, 0.1mg/ml BSA. |
| Storage and Stability | May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. |
| Overdigestion Assay | |
| No detectable change in the specific fragmentation pattern was observed after 160-fold overdigestion (10u/ug Ad2 DNA x 16 hours) with S1014-59. |
|
| Ligation/Recutting Assay | |
| After 50-fold overdigestion (3u/ug DNA x 17 hours) with S1014-59, more than 80% of the DNA fragments can be ligated in a reaction mixture containing 20–40u of T4 DNA Ligase/1ug of fragments and 10% PEG at a 5'-termini concentration of 0.06uM. More than 95% of these can be recut. |
|
| Labeled Oligonucleotide (LO) Assay | |
| No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide occured during incubation with 10 units of S1014-59 for 4 hours. |
|
| Blue/White Cloning Assay | |
| The mix of pUC57/HindIII, pUC57/Eco32I and pUC57/PstI digests was incubated with 10 units of SmiI for 16 hours. After religation and transformation, the background level of white colonies was <1%. |
|
| Incubation Temperature | |
| 30ºC. Incubation at 37ºC results in 70% activity. |
|
| Methylation Effects | |
| Dam, Dcm, CpG, EcoKI | Never overlaps; no effect. |
| EcoBI | May overlap; effect not determined |
|
| Stability during Prolonged Incubation | |
| A minimum of 0.1 unit of SmiI is required for complete digestion of 1ug of Ad2 DNA in 16 hours at 30°C |
| |
| Thermal Inactivation | |
| SmiI is inactivated by incubation at 65°C for 20min. |
|
| Digestion of Agarose-embedded DNA | |
| A minimum of 10 units of SmiI is required for digestion of 1ug of agarose-embedded Ad2 DNA in 16 hours. |
|
| Number of Recognition Sites in DNA | |
| Lambda | 0 |
| PhiX174 | 0 |
| pBR322 | 0 |
| pUC57 | 0 |
| pUC18/19 | 0 |
| pTZ19R/U | 0 |
| M13mp18/19 | 1 |
| Ad2 | 1 |
