Technical Data
M4692-60
MssI (PmeI)
250U
1250U
Molecular Biology Storage: -20CShipping: Blue Ice
5'-G T T T^A A A C-3'
3'-C A A A^T T T G-5'

Concentration:
5u/ul

Source:
Methylobacterium species Dd 5732

Unit Definition:
One unit is defined as the amount of Mssl required to digest 1ug of lambda DNA in 1 hour at 37C 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 37C), 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:
37C

Storage and Stability:
Aliquot and store at -20C. Aliquots are stable for at least 6 months at -20C. 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 65C 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 37C.

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 37C 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 37C for 1-16 hours.
Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.