| 5'-^ 7(N) G A A C (N)6 T C C (N)12–13^-3' |
|
| 3'-^12–13(N) C T T G (N)6 A G G (N)7 ^-5' |
|
| Catalog # | A1356 |
| Concentration | 2u/ul |
| Source | E.coli that carries the cloned aloIR gene from Acinetobacter lwoffi Ks4–8 |
| Buffer | 10mM Tris-HCl (pH 8.5), 10mM MgCl2, 100mM KCl and 0.1mg/ml BSA. Incubate at 30°C. Incubation at 37°C results in 20% 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 Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA and 50% glycerol. |
| Overdigestion Assay | No detectable change in the specific fragmentation pattern is observed after 10-fold overdigestion (5u/ug lambda DNA x 2 hours) with AloI (see Star Activity). |
|
| Ligation/Recutting Assay | After 10-fold overdigestion (0.6u/ug DNA x 17 hours) with AloI, more than 70% of the DNA fragments can be ligated at a 5'-termini concentration of 0.06uM. More than 80% of these can be recut. |
|
| Labeled Oligonucleotide (LO) Assay | No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of restriction endonuclease for 4 hours. |
|
| Blue/White Cloning Assay | The mix of pUC57/HindIII, pUC/PstI and pUC57/Eco32I digests was incubated with 10units of enzyme for 16 hours. After religation and transformation 0.6% of white colonies were detected. |
|
| Star Activity | An excess of enzyme (6u/ug lambda DNA x 2 hours) may result in star activity. |
|
| Methylation Effects | AloI does not cut GGA(N)6GTTm5C. Overlapping CG methylation may influence DNA cleavage. |
|
| Stability during Prolonged Incubation | A minimum of 0.1units of enzyme is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C. |
|
| Thermal Inactivation | Enzyme is inactivated by incubation at 65°C for 20min. |
|
| Number of Recognition Sites in DNA | |
|
| Lambda | 7 |
| PhiX174 | 0 |
| M13mp18/19 | 1 |
| pBR322 | 0 |
| pUC18/19 | 0 |
| pUC57 | 0 |
| pTZ19R/U | 1 |
| pBluescriptIIKS(-/+) | 1 |
| pBluescriptIISK(-/+) | 1 |
| pACYC177 | 0 |
| pACYC184 | 0 |
|
| Note | AloI produces double-strand cuts on both sides from the interrupted recognition site. Its unique feature is a degenerate cleavage point on the 3' side of the recognition sequence (12 or 13 nt away). Addition of SAM to the reaction mixture results in incomplete cleavage. |
|
| Unit Definition | One unit is defined as the amount of enzyme required to digest 1ug of lambda DNA in 1 hour at 30°C in 50ul of assay buffer. |
