MnlI
(Moraxella nonliquefaciens)
5’-C C T C (N)7 -3’
3’-G G A G (N)6-5’
Reaction Temperature: 37°C
Prototype: MnlI
Inactivation Temperature (20 min): 65°C
Reaction Buffer:
1x Medium Buffer: 10 mM Tris-HCl (pH 7.5 at 37°C), 10 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 100 µg/ml bovine serum albumin.
Unit Definition: One unit is the amount of enzyme required to completely digest 1 µg of lambda DNA in 1 hr in a total reaction volume of 50 µl.
Assay Conditions: 10 mM Tris-HCl (pH 7.5 at 37°C), 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol,1 µg of lambda DNA and 100 µg/ml bovine serum albumin. Incubation is at 37°C for 1 hr in a reaction volume of 50 µl.
Storage Buffer: 10 mM Tris-HCl (pH 7.5 at 22°C), 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 200 µg/ml bovine serum albumin and 50% (v/v)glycerol.
Storage Conditions: Store at –20°C.
Quality Control:
Non-specific Endonuclease: Incubation of 10 units of MnlI with 1 µg of lambda DNA at 37°C for 16 hrs (a 160-fold over-digestion) resulted in the same sharp characteristic banding pattern as the standard assay reaction, as determined by agarose gel electrophoresis.
3'-Exonuclease: 1.5, 3 and 6 units of MnlI and 0.08 µg (0.61 pmol of 3'-ends) of lambda/TaqI fragments (3'-labeled with T4 DNA Polymerase and [³H]dGTP and [³H]dCTP), incubated for 1 hr at 37°C resulted in 0.03 slope of %-end label released per unit of enzyme. Reaction volume 10 µl.
5'-Exonuclease/5'-Phosphatase: Incubation of 1.5, 3 and 6 units of MnlI with 0.16 µg (1.48 pmol of 5'-ends) of [5'-³²P]lambda / HaeIII fragments for 1 hr at 37°C resulted in a less than 0.16 slope of %-end label released per unit of enzyme. Reaction volume 10 µl.