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.
Product SourceAn E. coli strain that carries the cloned and modified SbfI gene from Streptomycesspecies Bf-61 (S.K. Degtyarev).
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
|Gel Loading Dye, Purple (6X)||25||6X|
Properties and Usage
Unit DefinitionOne unit is defined as the amount of enzyme required to digest 1μg of λ DNA 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 NEBuffersNEBuffer 1.1: 50%
NEBuffer 2.1: 25%
NEBuffer 3.1: 10%
CutSmart® Buffer: 100%
10 mM Tris-HCl
200 mM NaCl
1 mM DTT
0.1 mM EDTA
200 μg/ml BSA
pH 7.4 @ 25°C
Heat Inactivation80°C for 20 min
dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive
Quality Control AssaysThe 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.
- 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.
This 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@example.com.
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.
Gel Loading Dye, Purple (6X)
- 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.
- Is there a difference in cutting close to the ends between SbfI-HF and SbfI?
- What is the difference between SbfI-HF and SbfI?
- How does the level of star activity of SbfI-HF compare to SbfI?
- When should I choose the HF version of the enzyme?
- When is star activity a concern?
- Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
- Can the change in buffer preference of the HF enzyme be advantageous?
- Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended?
- 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?
- What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
- Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
- How can I access the old NEBuffer™ Activity Chart?
- 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?
- Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?
- 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?
- Why is my Restriction Enzyme not cutting DNA?
- Why do I see additional DNA bands on my gel after a restriction digest?
- Why do I see a DNA smear on an agarose 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?
- Alphabetized List of Recognition Specificities
- Average Fragment Size Generated By Endonuclease Cleavage
- Buffer and Diluent Formulation Table
- Compatible Cohesive Ends and Generation of New Restriction Sites
- Cross Index of Recognition Sequences
- Dam-Dcm and CpG Methylation
- Frequencies of Restriction Sites
- Time-Saver™ Qualified Enzymes
- Why Choose Recombinant Enzymes?
Usage Guidelines & Tips
- 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
- Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
- Double Digests
- Heat Inactivation
- Megabase Mapping
- 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
- Kanamori A, Sugita Y, Yuasa Y, Suzuki T, Kawamura K, Uno Y, Kamimura K, Matsuda Y, Wilson CA, Amores A, Postlethwait JH, Suga K, Sakakura Y. (2016). A Genetic Map for the Only Self-Fertilizing Vertebrate. G3 (Bethesda). Apr 7;6(4), 1095-106.
- Hou Y, Nowak MD, Mirré V, Bjorå CS, Brochmann C, Popp M. (2016). RAD-seq data point to a northern origin of the arctic-alpine genus Cassiope (Ericaceae). Mol Phylogenet Evol. . Feb;95, PubMedID: 152-60
- Demos TC, Kerbis Peterhans JC, Joseph TA, Robinson JD, Agwanda B, Hickerson MJ. (2015). Comparative Population Genomics of African Montane Forest Mammals Support Population Persistence across a Climatic Gradient and Quaternary Climatic Cycles. PLoS One. . Sep 22;10(9), e0131800.
- Lescak EA, Bassham SL, Catchen J, Gelmond O, Sherbick ML, von Hippel FA, Cresko WA. (2015). Evolution of stickleback in 50 years on earthquake-uplifted islands. Proc Natl Acad Sci U S A. Dec 29;112(52), E7204-12.
- Herrera S, Watanabe H, Shank TM. (2015). Evolutionary and biogeographical patterns of barnacles from deep-sea hydrothermal vents. Mol Ecol.. Feb;24(3), 673-89.
- Kemppainen P, Knight CG, Sarma DK, Hlaing T, Prakash A, Maung Maung YN, Somboon P, Mahanta J, Walton C. (2015). Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions, local adaptation and geographic structure. Mol Ecol Resour. . Sep;15(5), 1031-45.
- Gamble T, Coryell J, Ezaz T, Lynch J, Scantlebury DP, Zarkower D. (2015). Restriction Site-Associated DNA Sequencing (RAD-seq) Reveals an Extraordinary Number of Transitions among Gecko Sex-Determining Systems. Mol Biol Evol. . May;32(5), 1296-309.
- Hou Y, Nowak MD, Mirré V, Bjorå CS, Brochmann C, Popp M. (2015). Thousands of RAD-seq Loci Fully Resolve the Phylogeny of the Highly Disjunct Arctic-Alpine Genus Diapensia (Diapensiaceae). PLoS One. . Oct 8;10(10), e0140175.