| 5'-T G G^C C A-3' |
|
| 3'-A C C^G G T-5' |
|
| Catalog # | M4130 |
| Source | Micrococcus luteus Ng 16-122 |
| Concentration | 5u/ul |
| Unit Definition | One unit is defined as the amount of MlsI required to digest 1ug of lambda DNA dcm in 1 hour at 37°C in 50ul of reaction buffer. |
| Supplied With | R1625: Restriction Enzyme Buffer A, 10X: Supplied as a liquid in 33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA (pH 7.9 at 37°C). |
| R1625-04 | Restriction Enzyme Buffer E, 10X: Supplied as a liquid in 10mM Tris-HCl, pH 8.5, 10mM MgCl2, 100mM KCl and 0.1mg/ml BSA. Incubate at 37°C. |
| Storage and Dilution Buffer | Supplied as a liquid in 10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol. |
| Thermal Inactivation | MlsI is inactivated by incubation at 65°C for 20min. |
| Overdigestion Assay | |
| No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with MlsI. |
|
| Ligation/Recutting Assay | |
| After 50-fold overdigestion (3u/ug DNA x 17 hours) with MlsI, more than 90% of the DNA fragments can be ligated at a 5'-termini concentration of 0.9uM. More than 95% of these can be recut. |
|
| Labeled Oligonucleotide (LO) Assay | No detectable degradation of single- stranded or double-stranded labeled oligonucleotide was observed after incubation with 10 units of MlsI for 4 hours. |
|
| Stability during Prolonged Incubation | |
| A minimum of 0.5units of MlsI is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C. |
|
| Number of Recognition Sites in DNA | |
| Lambda | 18 |
| PhiX174 | 0 |
| M13mp18/19 | 1 |
| pBR322 | 1 |
| pUC18/19 | 0 |
| pUC57 | 0 |
| pTZ19R/U | 0 |
|
| Digestion of Agarose-embedded DNA | A minimum of 20 units of MlsI is required for digestion of 1ug of agarose-embedded lambda DNA in 16 hours. |
|
| Recommended Protocol for Digestion | |
| Add | |
| Nuclease free water | 16ul |
| R1625-04 | 2ul |
| DNA (0.5-1ug/ul) | 1ul |
| MlsI | 0.5-2ul |
| Mix gently, spin down for a few seconds. |
| Incubate at 37ºC for 1-2 hours. |
|
| Protocol for Digestion of PCR products directly after amplification | |
| Add | |
| PCR Reaction Mixture | 10ul (~0.1-0.5ug of DNA) |
| Nuclease free water | 18ul |
| R1625-04 | 2ul |
| MlsI | 1-2ul |
| Mix gently, spin down for a few seconds. |
| Incubate at 37ºC for 1-16 hours. |
|
| Storage and Stability | |
| May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. |
