MlsI (BalI)

Blue Ice

Molecular Biology Grade

5'-T G G^C C A-3'
3'-A C C^G G T-5'

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. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months after receipt 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.