Product Source

An E.coli strain that carries the cloned NmeAIII gene from Neisseria meningitidis 2491 (Achtman, M.)

Reagents Supplied

The following reagents are supplied with this product:

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

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to digest 1 µg of ΦX174 RF I DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

Reaction Conditions

1X CutSmart® Buffer
Supplement with 80 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: 10%
NEBuffer 2.1: 10%
NEBuffer 3.1: 10%
CutSmart® Buffer: 100%

Diluent Compatibility

Storage Temperature


Storage Conditions

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

Heat Inactivation

65°C for 20 min

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive


  1. Overdigestion with > 5 units of NmeAIII per µg of DNA is not recommended.
  2. Requires S-adenosylmethionine for optimal activity (supplied with enzyme)
  3. NmeAIII requires two copies of its recognition sequence for cleavage to occur. Thus, the single NmeAIII site in pUC19 is resistant to cleavage. A 10-fold overdigestion cuts less than half of the DNA.
  4. The cleavage point may shift one base pair depending on the DNA sequence context between the recognition site and the position of cleavage. For a given sequence, generally one cut site will predominate.
  5. Significant cleavage occurs on ice and at 25°C. 
  6. NmeAIII produces a stable partial digestion pattern even with excess enzyme. 1 unit is defined as the amount of enzyme required to produce this stable partial digestion pattern.
  7. 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. How can I search for a restriction enzyme by sequence, overhang or name?
  2. How should I stop my restriction digest?
  3. How stable is a particular restriction enzyme?
  4. When should I choose the HF version of an enzyme?
  5. When is star activity a concern?
  6. What does it mean to be Time-Saver™ qualified?
  7. What are the advantages of using a RE-Mix Restriction Enzyme Master Mix?
  8. How should I set up a restriction digest?
  9. I don't see any cleavage after my restriction digest. What factors can interfere with cleavage?
  10. How can I generate a restriction enzyme site map for my sequence?
  11. What information is available in the Restriction Enzyme Database (REBASE)?
  12. Is extended digestion (incubation times > 1 hour) recommended?
  13. Do degenerate recognition sites need to be palindromic?
  14. My enzyme is no longer Time-Saver™ qualified. What happened?
  15. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  16. How can I access the old NEBuffer™ Activity Chart?
  17. 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?
  18. Why is my Restriction Enzyme not cutting DNA?
  19. Why do I see a DNA smear on an agarose gel after a restriction digest?
  20. Why do I see additional DNA bands on my gel after a restriction digest?
  21. 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

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.
  • 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.

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.