New England Biolabs, R0163L, MnlI

MnlI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10161943. Related Categories Restriction Endonucleases H M,, Time-Saver Qualified Restriction Enzymes Applications Fast Cloning: Accelerate your cloning workflows with reagents from NEB,, Restriction Enzyme Digestion Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 37°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: 75% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 50% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent B Storage Buffer 10 mM Tris-HCl 300 mM NaCl 1 mM DTT 0.1 mM EDTA 500 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Not Sensitive FAQ Q: How do you recommend using MnlI? A: 1. MnlI works best on DNA that has been washed or dialyzed. 2. Don't digest for more than 4 hours. 3. If cleavage is incomplete, use more enzyme. Q: Is there variability in the cleavage site for MnlI? A: There appears to be a wobble effect that imitates star activity, and represents a variable cleavage site. MnlI always leave a single-base 3´ overhang. The majority of sites under our unit definition conditions will cut (7/6) from the recognition site, but some sites have been reported to cut at (7/8) from the recognition site. Q: Has the recognition or cleavage site been revised for MnlI? A: The cleavage site of MnlI has been revised. Brinkly, P., Bausista, DS, and Graham, FL (1991) Gene: 100. 267-268. Q: Does MnlI cleave supercoiled DNA? A: Two times the amount of MnlI is required to digest puc19. Q: What is the activity of MnlI at 25°C? A: It is 25-50% active. Q: Does MnlI cleave ssDNA? A: MnlI cleaves single stranded DNA at least 50% slower. Q: Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer? A: NEB's Time-Saver™ enzymes have the benefit of working fast (5-15 minutes), but are also designed and qualified to withstand overnight digestions without degradation of DNA. Q: I tested your restriction enzyme on the substrate DNA recommended by NEB, and it appears to be active, however it does not digest my DNA. What could be the reason? A: The quality and cleanliness of the DNA are major factors that can affect enzyme activity. Restriction Enzymes active in rCutSmart® Buffer, but not as active in NEBuffer r2.1 and/or r3.1 (our higher salt buffers), can be inhibited by salt in the reaction. DNA purification procedures that use spin columns can result in DNA solutions with significant levels of salt that can carry over into the reaction and inhibit enzyme activity. To prevent this, we recommend that the DNA solution added to the reaction be no more than 25% of the total reaction volume. This can be attained by adjusting the total reaction volume accordingly. Q: Is this enzyme sensitive to dam, dcm or mammalian CpG methylation? A: No. This enzyme is not sensitive to dam, dcm, or mammalian CpG methylation. For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price.