Product Source

An E. coli strain that carries the MmeI gene from Methylophilus methylotrophus.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
S-adenosylmethionine (SAM)-2032 mM
CutSmart® Buffer-2010X

Properties and Usage

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 CutSmart® Buffer
Supplement with 50 μM S-adenosylmethionine (SAM)
Incubate at 37°C

1X CutSmart® Buffer:
50 mM Potassium Acetate
20 mM Tris-acetate
10 mM Magnesium Acetate
100 μg/ml BSA
pH 7.9 @ 25°C

Activity in NEBuffers

NEBuffer 1.1: 50%
NEBuffer 2.1: 100%
NEBuffer 3.1: 50%
CutSmart® Buffer: 100%

Diluent Compatibility

Storage Temperature


Storage Conditions

10 mM Tris-HCl
300 mM NaCl
0.1 mM EDTA
500 μg/ml BSA
50% Glycerol
1 mM DTT
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 min

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Blocked by Overlapping


  1. Excess MmeI blocks cleavage.
  2. Reactions using MmeI should be done at or near stoichiometric concentrations.
  3. Complete cleavage occurs within 15 minutes at 37°C.
  4. Significant cleavage occurs on ice and at 50°C.
  5. MmeI activity is inhibited by high ionic strength (> 200 mM).
  6. SAM must be present in a reaction at a concentration of 50 µM or higher for optimal cleavage.
  7. MmeI prefers two copies of its recognition sequence for cleavage to occur, with limited cleavage on plasmids with a single site.
  8. Based on the stability of the enzyme in the reaction, incubations longer than 1 hr will not result in improved digestion, unless additional enzyme is added. Please refer to Restriction endonuclease survival in a reaction for more information regarding this topic.


  1. Is there any variability in the distance of cutting from the MmeI cut site which is indicated as 20/18?
  2. I am observing partial digestion when using MmeI. What could be the reason?
  3. Is MmeI sensitive to CpG methylation?
  4. Why can a cleaved MmeI site religate but not be re-cut?
  5. Is MmeI cutting exact or is the spacing variable?
  6. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  7. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  8. How can I access the old NEBuffer™ Activity Chart?
  9. 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?
  10. Why is my Restriction Enzyme not cutting DNA?
  11. Why do I see a DNA smear on an agarose gel after a restriction digest?
  12. Will incubation overnight improve my digest?
  13. Why do I see additional DNA bands on my gel after a restriction digest?
  14. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?


  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes
  3. Time-Saver Protocol for Restriction Enzyme Digests

Selection Charts

Feature Articles

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Ligation and Recutting (Terminal Integrity):
    After an over-digestion of DNA with a restriction endonuclease the percentage of the DNA fragments ligated with T4 DNA ligase and the percentage that can be recut are determined by agarose gel electrophoresis.
  • Non-Specific DNase Activity (16 hour):
    The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]


The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.