High Fidelity Enzymes

Product Update: A free vial of Gel Loading Dye, Purple (6x)  is now included with all HF restriction enzymes.

cloned at neb recombinant engineered timesaver 5min incubation temp heat inactivation cpg star activity blue white
Catalog #SizeConcentrationPriceQtyAdd to Cart
R3131S1,000 units20,000 units/ml$66.00Add to Cart
R3131L5,000 units20,000 units/ml$267.00Add to Cart
R3131M5,000 units100,000 units/ml$267.00Add to Cart
High-Fidelity (HF®) Restriction Endonucleases,
Restriction Endonucleases: N-O,
Time-Saver™ Qualified Restriction Enzymes
Restriction Enzyme Digestion


High Fidelity (HF®) Restriction Enzymes have 100% activity in CutSmart™ Buffer; single-buffer simplicity means more straightforward and streamlined sample processing. HF enzymes also exhibit dramatically reduced star activity. HF enzymes are all Time-Saver qualified and can therefore cut substrate DNA in 5-15 with the flexibility to digest overnight without degradation to DNA. Engineered with performance in mind, HF restriction enzymes are fully active under a broader range of conditions, minimizing off-target products, while offering flexibility in experimental design.

NEB extensively performs quality controls on all standard and high-fidelity (HF®) restriction enzymes. Examples of nuclease contamination studies for some of our HF restriction enzymes are shown below.

Restriction Enzyme Competitor Study: Nuclease Contamination

EcoRI, NotI, and BamHI from multiple suppliers were tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3’overhang or 5’ overhang containing oligonucleotides. The percent degradation is determined by capillary electrophoresis and peak analysis. The resolution is at the single nucleotide level.

Product Source

An E. coli strain that carries the cloned and modified NheI gene from Neisseria mucosa heidelbergensis (ATCC 25999)

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
CutSmart® Buffer-2010X
Gel Loading Dye, Purple (6X)256X

Properties and Usage

Unit Definition

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

Reaction Conditions

1X CutSmart® Buffer
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: 100%
NEBuffer 2.1: 25%
NEBuffer 3.1: 10%
CutSmart® Buffer: 100%

Diluent Compatibility

Storage Temperature


Storage Conditions

10 mM Tris-HCl
250 mM NaCl
1 mM DTT
0.1 mM EDTA
200 μg/ml BSA
50% Glycerol
0.15% Triton® X-100
pH 7.4 @ 25°C

Heat Inactivation

80°C for 20 min

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Blocked by Some Combinations of Overlapping


  1. NheI-HF® has the same specificity as NheI (NEB #R0131), but it has been engineered for reduced star activity.
  2. Cleaves to leave a 5´ CTAG extension which can be efficiently ligated to DNA fragments generated by AvrII, SpeI or XbaI.
  3. Activity inhibited by salt concentrations > 100 mM.


  1. Is there a difference in cutting close to the ends between NheI-HF and NheI?
  2. What is the difference between NheI-HF and NheI?
  3. Is there any difference in the methylation sensitivity between NheI-HF and NheI?
  4. How does the level of star activity of NheI-HF compare to NheI?
  5. Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
  6. When should I choose the HF version of an enzyme?
  7. When is star activity a concern?
  8. When should I choose the High Fidelity (HF®) version of the enzyme?
  9. Can the change in buffer preference of the HF enzyme be advantageous?
  10. What does it mean to be Time-Saver™ qualified?
  11. How is the improvement in fidelity of HF restriction endonucleases quantitated?
  12. What is the Fidelity Index (FI)?
  13. What does HF® refer to following the name of a restriction enzyme?
  14. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  15. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  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. Is Gel Loading Dye, Purple (6X) or Gel Loading Dye, Purple (6X), no SDS compatible with other DNA binding dyes such as SYBR® and GelRed™ during gel electrophoresis?
  19. Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?
  20. Why is my Restriction Enzyme not cutting DNA?
  21. Why do I see a DNA smear on an agarose gel after a restriction digest?
  22. Why do I see additional DNA bands on my gel after a restriction digest?
  23. 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

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.
  • Blue-White Screening (Terminal Integrity):
    A sample of DNA vector linearized with a 10-fold excess of a restriction endonuclease, religated and transformed into an E. coli strain expressing the LacZ beta fragment gene results in less than 1% white colonies.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • 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.