New England Biolabs, R0637L, MmeI

MmeI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10263881. Related Categories Restriction Endonucleases H M,, Time-Saver Qualified Restriction Enzymes Applications Restriction Enzyme Digestion Specification Unit Definition One unit is defined as the amount of MmeI required to digest 1 µg of ΦX174 RF I DNA in 1 hour at 37°C in 50 μl of reaction buffer. 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: 50% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 50% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent B Storage Buffer 10 mM Tris-HCl 300 mM NaCl 0.1 mM EDTA 500 µg/ml Recombinant Albumin 50% Glycerol 0.32 mM S-adenosylmethionine (SAM) 1 mM DTT pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Blocked by Overlapping FAQ Q: Is there any variability in the distance of cutting from the MmeI cut site which is indicated as 20/18? A: It is helpful to think of MmeI as placing the endonuclease at a (relatively) fixed distance from the recognition site, rather than the enzyme counting the bases between the recognition site and the position of cutting. The observation is that MmeI cuts predominately at 20/18 and 21/19, with approximately a 60%/40% ratio. There is also minor cutting at 19/17 (roughly a few percent). Individual sites can be cut at mostly one distance or can be cut at a mixture of distances (20/18 and 21/19); we believe this depends on the structure of the DNA between the recognition site and the cut site. Q: I am observing partial digestion when using MmeI. What could be the reason? A: There are several reasons why MmeI may not be cutting to completion:1. MmeI is sensitive to salt in the reaction and any salt carry-over from a DNA prep may inhibit the enzyme. Test the reaction on a commercial DNA to determine if this is the problem. 2. MmeI requires SAM for efficient cleavage, and SAM is extremely labile. Try to use a fresh tube of SAM as well as commercial DNA to see if this is the problem. 3. MmeI does not have much turn-over as an enzyme, unlike common Type IIP restriction enzymes. Thus it is important to have a 1:1 ratio of MmeI molecules to the number of sites to be cut in the reaction. This can be particularly important for short DNAs, since 1 microgram of a 100bp PCR product containing a single MmeI site represents approximately 10-fold more sites per microgram than the PhiX174 substrate used for MmeI unit definition. 4. MmeI requires two binding sites for efficient cleavage, and digestion of plasmids with a single site results in partial digestion of approximately 70% cutting. As MmeI requires 2 sites, addition of a double-strand oligo containing an MmeI site may help improve cleavage. The ds oligo should have 8 to 12 bp of flanking sequence on either side of the MmeI recognition sequence, but need not extend all the way to the point of cleavage. Q: Is MmeI sensitive to CpG methylation? A: MmeI is blocked by overlapping CpG methylation if the middle C, or the 3’ C, is followed by a G, resulting in CpG methylation of C. For example, the sequence TCCGAC, or the sequence TCCRACG, will not be cut by MmeI if CpG modification methylates the underlined Cytosine residue. For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Q: Why can a cleaved MmeI site religate but not be re-cut? A: MmeI is an unusual enzyme in that it has both restriction and methylation properties. Because DNA cleavage does not break the recognition site, the enzyme can methylate its recognition site following cleavage. Once the site is methylated, it can not be subsequently cut after being ligated. Q: Is MmeI cutting exact or is the spacing variable? A: MmeI cuts predominantly at 20/18, but can occasionally cut one base further or closer to the recognition site at 21/19 or 19/17. 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: Will incubation overnight improve my digest? A: MmeI does not need to be digested overnight, as complete cleavage will occur in 15 minutes. Long incubations are generally not helpful in achieving complete cutting, as the enzyme can and will methylate its site over time, which will prevent cleavage. Q: Does MmeI require SAM for activity? A: Yes, S-adenosylmethionine (SAM) is required for optimal activity (SAM is supplied in the enzyme formulation as of October 2020). If you are unsure whether SAM is in the enzyme formulation and you have a vial of SAM from NEB, it is fine to add SAM to the reaction per previous guidelines. 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.