New England Biolabs, M0250L, Mung Bean Nuclease

Related Categories Exonucleases and Non-specific Endonucleases,, RNA Modification Applications Blunting Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of M13mp18 single-stranded DNA to fragments less than 1 kb in length in a total reaction volume of 80 μl in 1X Mung Bean Nuclease Reaction Buffer when incubated for 15 minutes at 37°C. Reaction Conditions 1X Mung Bean Nuclease Reaction Buffer Incubate at 30°C 1X Mung Bean Nuclease Reaction Buffer 30 mM NaCl 50 mM sodium acetate 1 mM ZnSO4 (pH 5 @ 25°C) Storage Buffer 10 mM sodium acetate 0.1 mM Zinc Acetate 1 mM Cysteine 50% Glycerol 0.001% Triton® X-100 pH 5 @ 25°C Molecular Weight Theoretical: 39 kDa FAQ Q: Should I use Mung Bean Nuclease or DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210)? A: Mung Bean Nuclease must be used if the goal is to chew back a 5' extension. DNA Polymerase I, Large (Klenow) Fragment will fill in a 5' extension and it will chew back a 3' extension. Mung Bean Nuclease can be used to chew back a 3' extension, but DNA Polymerase I, Large (Klenow) Fragment may be a slightly better choice due to Mung Bean Nuclease's ability to act at nicks and degrade ends as they "breathe". Note: A 3' overhang can not be filled in. Q: Is the Mung Bean Nuclease active in other NEBuffers? A: Mung Bean Nuclease is also fully active rCutSmart® Buffer. The enzyme may also be used in NEBuffers 1, 2, 4, r1.1, r2.1 however the reaction time should be doubled to 1 hour at 30°C when these buffers are used. Activity is poor in NEBuffers 3 and r3.1. Q: Can Mung Bean Nuclease be heat inactivated? A: DO NOT ATTEMPT TO HEAT INACTIVATE ! Although Mung Bean Nuclease can be inactivated by heat, this is not recommended because the DNA begins to "breathe" before the Mung Bean Nuclease is inactivated and undesirable degradation occurs at breathing sections. Purify DNA by phenol/chloroform extraction and ethanol precipitation or spin column purification. Q: Does Zn2+ need to be added to a Mung Bean Nuclease reaction? A: It is no longer necessary to supplement Mung Bean Nuclease reactions with Zn2+. The zinc acetate in the storage buffer fulfills the Zn2+ requirement of the enzyme even after dilution in a reaction. Q: Will Mung Bean Nuclease degrade double stranded DNA? A: When used at temperatures higher than the 30°C recommended, or for longer periods, degradation of the substrate DNA will result. Further, since the endonucleic activity is known to occur at 'bubbles' on the DNA helix, which transiently form along the length of the molecule, substrates which are already nicked, or are of high AT content are more susceptible to this endonucleic activity.