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.
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.
- 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.
- Cleavage of mammalian genomic DNA is blocked by CpG methylation.
- 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 does it mean to be Time-Saver™ qualified?
- 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?
- How can I access the old Double Digest Finder?
- 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?
Usage Guidelines & Tips
- Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
- Activity of Restriction Enzymes in a Q5®, Taq or Phusion PCR Mix
- Cleavage Close to the End of DNA Fragments
- Cleavage of Supercoiled DNA
- Dam and Dcm Methylases of E. coli
- Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
- Double Digests
- Effects of CpG Methylation on Restriction Enzyme Cleavage
- 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
- Single Letter Codes
- Star Activity
- Traditional Cloning Quick Guide