DescriptionEcoRI has a High Fidelity version EcoRI-HF® (NEB #R3101).
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 minutes 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 EcoRI gene from E. coli RY13 (R.N. Yoshimori).
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
Properties and Usage
Unit DefinitionOne unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
1X NEBuffer EcoRI
Incubate at 37°C
1X NEBuffer EcoRI:
100 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
0.025% Triton® X-100
pH 7.5 @ 25°C
Activity in NEBuffersNEBuffer 1.1: 25%
NEBuffer 2.1: 100%
NEBuffer 3.1: 50%
CutSmart™ Buffer: 50%
10 mM KPO4
300 mM NaCl
0.1 mM EDTA
200 μg/ml BSA
10 mM DTT
0.15% Triton® X-100
pH 7.5 @ 25°C
Heat Inactivation65°C for 20 min
dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Blocked by Some Combinations of Overlapping
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.
- 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.
- 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.
- May exhibit star activity in NEBuffer 2.1 or CutSmart Buffer.
- What is Star Activity and how can it be avoided?
- 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?
- My restriction enzyme used to work well in the old NEBuffer but the new Performance chart indicates it has lower activity even though the only difference is the addition of BSA and removal of DTT to the new buffers. Why?