| 5'-G T T T^A A A C-3' |
|
| 3'-C A A A^T T T G-5' |
|
| Catalog # | M4692-60 |
| Concentration | |
| 5u/ul |
| Source | Methylobacterium species Dd 5–732 |
| Unit Definition | One unit is defined as the amount of Mssl required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer. |
| Diluent Buffer | 10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol. |
| Storage Buffer | 10mM Tris-HCl, pH 7.5, 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% glycerol. |
| Supplied with | R1625-Restriction Enzyme Buffer A, 10X: Dilute to 1X for use. 1X buffer composition is 33mM Tris-acetate (pH 7.9 at 37°C), 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA. |
| R1625-04 Restriction Enzyme Buffer E, 10X | Dilute to 1X for 100% Mssl digestion. 10mM Tris-HCl, pH 7.5, 10mM MgCl2 and 0.1mg/ml BSA. |
| Incubation Temperature | 37°C |
| Storage and Stability | 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. |
| Overdigestion Assay | |
| No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with Mssl. |
|
| Ligation/Recutting Assay | |
| After 50-fold overdigestion (0.6u/ug DNA x 17 hours) with Mssl, more than 90% of the DNA fragments can be ligated in a mixture containing 20-40u of T4 ligase/ 1ug of fragments and 10% PEG at a 5'-termini concentration of 0.09uM. More than 90% 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 Mssl for 4 hours. |
|
| Blue/White Cloning Assay | |
| A mixture of pUC57/Hindlll, pUC57/Pstl and pUC57/Eco321 digests was incubated with 10 units of Mssl for 16 hours. After reeligation and transformation, the background level of white colonies was <1%. |
|
| Thermal Inactivation | |
| Mssl is inactivated by incubation at 65°C for 20min. |
|
| Stability during Prolonged Incubation | |
| A minimum of 5 units of Mssl is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C. |
|
| Number of Recognition Sites in DNA | |
| Lambda | 2 |
| PhiX174 | 0 |
| M13mp18/19 | 0 |
| pBR322 | 0 |
| pUC18/19 | 0 |
| pUC57 | 0 |
| pTZ19R/U | 0 |
|
| Protocol for Digestion | |
| Add | Nuclease free water: 16ul |
| R1625-04 | 2ul |
| DNA (0.5-1ug/ml) | 1ul |
| M4692-60 | 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 |
| R1625-04 | 2ul |
| M4692-60 | 1-2ul |
| Mix gently and spin down for a few seconds. |
| Incubate at 37ºC for 1-16 hours. |
