Mnl I

Blue Ice

Molecular Biology Grade

5'...C C T C (N)7↓...3'
3'...G G A G (N)6↑...5'

Source:
E. coli with cloned mnlIR gene from Moraxells nonliquefaciens

Concentration:
10units/ul

Unit Definition:
One unit is defined as the amount of Mnl I required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer.

Incubation Temperature:
37°C

Form:
Supplied as a liquid in 10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA, 50% glycerol.

Dilution Buffer:
Dilute in the same buffer as Form above.

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
R1625-02: Restriction Enzyme Buffer C, 10X
Dilute to 1X. 1X buffer compostion is 10mM Tris-HCl pH 7.5, 10mM MgCl2, 50mM sodium chloride. 0.1mg/ml BSA

Enyme Properties:
Methylation Effects:
Dam: Never overlaps - no effect
Dcm: Never overlaps - no effect
CpG: May overlap - no effect
EcoKI: Never overlaps - no effect
EcoBI: May overlap - Blocked

Stability during Prolonged Incubation:
A minimum of 0.5units of Mnl I are required for complete digestion of 1ug of DNA in 16 hours at 37°C.

Thermal Inactivation:
Mnl I is inactivated by incubation at 65°C for 20 minutes.

Number of Recognition Sites in DNA:
Lambda: 262
PhiX174: 34
pBR322: 26
pUC57: 14
pUC18/19: 13
pTZ19R/U: 12
M13mp18/19: 61

Notes:
• Mnl I produces DNA fragments that have a single base 3’-extension which are more difficult to ligate than blunt-ended fragments.
• Mnl I may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, heat the digested DNA in the presence of SDS prior to electrophoresis.

Quality Control:

Overdigestion Assay:
No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with Mnl I.

Ligation/Recutting Assay:
The ligation and recleavage assay was replaced with LO test after validating experiment showed LO test ability to trace nuclease and phosphatase activities with sensitivity that is hihgher than L/R by a factor of 100.

Labeled Oligonucleotide (LO) Assay:
No detectable degradation of a single-stranded or double-stranded labeled oligonucleotide was observed after incubation with 10units of Mnl I for 4 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.