5'-G T T T^A A A C-3' 3'-C A A A^T T T G-5'
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
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.
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.
Source
Methylobacterium species Dd 5–732
Important Note
This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
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