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High-Fidelity (HF®) restriction enzymes have the same specificity as native enzymes, but have been engineered for significantly reduced star activity and performance in a single buffer (CutSmart® Buffer). All HF-restriction enzymes come with Gel Loading Dye, Purple (6X) . Enjoy the enhanced performance and added value of our engineered enzymes at the same price as the native enzyme:
- Engineered for improved performance
- 100% activity in CutSmart Buffer
- Time-Saver™ qualified for digestion in 5-15 minutes
- Reduced star activity
- Supplied with 1 vial of Gel Loading Dye, Purple (6X)
Featured VideosView Video Library
Reduce Star Activity with High-Fidelity Restriction Enzymes
Time-Saver Protocol for Restriction Enzyme Digests
NEB TV Ep. 15 – Applications of Restriction Enzymes
CutSmart Restriction Enzyme Buffer
Restriction Enzyme Digest Protocol: Cutting Close to DNA End
Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel
Why is My Restriction Enzyme Not Cutting DNA?
Restriction Enzyme Digest Problem: Too Many DNA Bands
Double Digestion with NEBcloner
DescriptionHigh 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.
Product SourceAn E. coli strain that carries the cloned and modified NheI gene from Neisseria mucosa heidelbergensis (ATCC 25999)
Properties & Usage
Unit DefinitionOne 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.
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 NEBuffersCutSmart® Buffer: 100%
NEBuffer™ 1.1: 100%
NEBuffer™ 2.1: 25%
NEBuffer™ 3.1: 10%
10 mM Tris-HCl
250 mM NaCl
1 mM DTT
0.1 mM EDTA
200 µg/ml BSA
0.15% Triton® X-100
pH 7.4 @ 25°C
Heat Inactivation80°C for 20 min
dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Blocked by Some Combinations of Overlapping
- NheI-HF® has the same specificity as NheI (NEB #R0131), but it has been engineered for reduced star activity.
- Cleaves to leave a 5´ CTAG extension which can be efficiently ligated to DNA fragments generated by AvrII, SpeI or XbaI.
- Activity inhibited by salt concentrations > 100 mM.
- Blocked by some combinations of overlapping CpG methylation.
Protocols, Manuals & Usage
Usage & Guidelines
- Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
- Activity of Restriction Enzymes in PCR Buffers
- Cleavage Close to the End of DNA Fragments
- Cleavage of Supercoiled DNA
- Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
- Double Digests
- Effects of CpG Methylation on Restriction Enzyme Cleavage
- Heat Inactivation
- NEBuffer Activity/Performance Chart with Restriction Enzymes
- Optimizing Restriction Endonuclease Reactions
- Reduced Star Activities of HF® Enzymes
- Restriction Endonucleases - Survival in a Reaction
- Restriction Enzyme Diluent Buffer Compatibility
- Restriction Enzyme Tips
- Single Letter Codes
- Star Activity
- Traditional Cloning Quick Guide
Tools & Resources
- Alphabetized List of Recognition Specificities
- Buffer and Diluent Formulation Table
- Compatible Cohesive Ends and Generation of New Restriction Sites
- Dam-Dcm and CpG Methylation
- Frequencies of Restriction Sites
- Recleavable Filled-in 5' Overhangs
- Time-Saver™ Qualified Enzymes
- Why Choose Recombinant Enzymes?
FAQs & Troubleshooting
- Is there a difference in cutting close to the ends between NheI-HF and NheI?
- What is the difference between NheI-HF and NheI?
- Is there any difference in the methylation sensitivity between NheI-HF and NheI?
- How does the level of star activity of NheI-HF compare to NheI?
- Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
- When should I choose the HF version of an enzyme?
- When is star activity a concern?
- When should I choose the High Fidelity (HF®) version of the enzyme?
- Can the change in buffer preference of the HF enzyme be advantageous?
- What does it mean to be Time-Saver™ qualified?
- How is the improvement in fidelity of HF restriction endonucleases quantitated?
- What is the Fidelity Index (FI)?
- What does HF® refer to following the name of a restriction enzyme?
- Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
- 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?
- 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?
- Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?
- Why is my Restriction Enzyme not cutting DNA?
- Why do I see a DNA smear on an agarose gel after a restriction digest?
- Why do I see additional DNA bands on my gel after a restriction digest?
- How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?
- Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)?
Quality, Safety & Legal
Quality Assurance StatementQuality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
SpecificationsThe 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]
Certificate Of AnalysisThe Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Safety DataSheetsThe following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
Gel Loading Dye, Purple (6X)
Legal and DisclaimersThis product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
The supporting documents available for this product can be downloaded below.