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 PvuI gene from Proteus vulgaris (ATCC 13315).
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 1 µg of pXba 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: 25%
NEBuffer 2.1: 100%
NEBuffer 3.1: 100%
CutSmart® Buffer: 100%
10 mM Tris-HCl
300 mM NaCl
1 mM DTT
0.1 mM EDTA
500 μg/ml BSA
pH 7.4 @ 25°C
dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Blocked
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.
- Cleavage of mammalian genomic DNA is blocked by CpG methylation.
- For enzymes that cannot be heat-inactivated, we recommend using a column for cleanup (such as the Monarch® PCR & DNA Cleanup Kit), or running the reaction on an agarose gel and then extracting the DNA (we recommend Monarch Gel Extraction Kit), or performing a phenol/chloroform extraction.
- How can I search for a restriction enzyme by sequence, overhang or name?
- How should I stop my restriction digest?
- How stable is a particular restriction enzyme?
- When should I choose the HF version of the enzyme?
- When is star activity a concern?
- What are the advantages of using a RE-Mix Restriction Enzyme Master Mix?
- How should I set up a restriction digest?
- I don't see any cleavage after my restriction digest. What factors can interfere with cleavage?
- How can I generate a restriction enzyme site map for my sequence?
- What information is available in the Restriction Enzyme Database (REBASE)?
- Is extended digestion (incubation times > 1 hour) recommended?
- Do degenerate recognition sites need to be palindromic?
- 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?
- 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?
- What does it mean to be Time-Saver™ qualified?
- 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?
- Why is my Restriction Enzyme not cutting DNA?
- How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?