|Molecular Biology||Storage: -20°CShipping: Blue Ice|
5'-G T T T^A A A C-3'
3'-C A A A^T T T G-5'
Methylobacterium species Dd 5–732
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.
10mM Tris-HCl, pH 7.5, 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
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.
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.
No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with Mssl.
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%.
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:
Protocol for Digestion:
Add: Nuclease free water: 16ul
DNA (0.5-1ug/ml): 1ul
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
Mix gently and spin down for a few seconds.
Incubate at 37ºC for 1-16 hours.