| 5'-G T C T C (N)1^-3' |
|
| 3'-C A G A G (N)5^-5' |
|
| Catalog # | A1372-10 |
| Source | Acinetobacter lwoffi RFL26 |
| Concentration | 10u/ul |
| Unit Definition | One unit is defined as the amount of Alw26I required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer. |
| Incubation Temperature | 37°C |
| Diluent Buffer | 10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol. |
| Storage Buffer | Supplied as a liquid in 10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA and 50% glycerol. |
| Restriction Enzyme Buffer A, 10X | R1625: Supplied as a liquid in 33mM Tris-acetate, pH 7.9, 10mM magnesium acetate, 66mM potassium acetate and 0.1mg/ml BSA. Incubate at 37°C. Supplied to simplify buffer selection for double digests. |
| Storage and Stability | May be stored at 4°C for short-term only. For long-term storage, aliquot and store at -20°C. Aliquots are stable for at least 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. |
| Quality Control | |
| Overdigestion Assay | |
| No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with Alw26I. |
|
| Ligation/Recutting Assay | |
| After 50-fold overdigestion (3u/ug DNA x 17 hours) with Alw26I more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.1uM. More than 95% 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 10units of Alw26I for 4 hours. |
|
| Methylation Effects | |
| Alw26I does not cut GAGAm5C. Overlapping CpG methylation may influence DNA cleavage. |
|
| Stability during Prolonged Incubation | |
| A minimum of 0.2units of Alw26I is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C. |
|
| Thermal Inactivation | |
| Alw26I is inactivated by incubation at 65°C for 20min. |
|
| Number of Recognition Sites in DNA | |
| Lambda | 37 |
| PhiX174 | 4 |
| M13mp18/19 | 5 |
| pBR322 | 3 |
| pUC18/19 | 4 |
| pUC57 | 4 |
| pTZ19R/U | 2 |
|
| Protocol for Digestion | |
| Add | |
| Nuclease free water | 16ul |
| R1625 | 2ul |
| DNA (0.5-1ug/ml) | 1ul |
| A1372-10 | 0.5-2ul |
| Mix gently and spin down for a few seconds. Incubate at 37ºC for 1-16 hours. |
|
| Protocol for Digestion Directly after Amplification | |
| Add | |
| PCR Reaction Mixture | 10ul (~0.1-0.5ug of DNA) |
| Nuclease free water | 18ul |
| R1625 | 2ul |
| A1372-10 | 1-2ul |
| Mix gently and spin down for a few seconds. |
| Incubate at 37ºC for 1-16 hours. |
