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Restriction Endonucleases >
High Fidelity (HF) Restriction Enzymes >
EcoRI-HF™ |
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Prices are in US dollars and valid only for US orders.
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Recognition Site:
isoschizomers | compatible ends | single letter code
Description:
New England Biolabs provides customers with high quality tools for a wide range of molecular biology applications. As part of our ongoing commitment to the study and improvement of restriction enzymes, we are pleased to introduce a line of restriction enzymes that have been engineered for maximum performance, convenience and flexibility. These engineered enzymes have the same specificity as their established counterparts with the benefit of reduced star activity. In addition, all of these engineered enzymes work optimally in NEBuffer 4, which has the highest level of enzyme compatibility, and will simplify double digest reactions. They are all Time-Saver qualified, and digest substrate DNA in five minutes. In order to distinguish these engineered enzymes, the letters –HF™ have been added to the restriction enzyme name and they are packaged in unique purple-capped tubes.
Source:
An E. coli strain that carries the cloned and modified (K62E) EcoRI gene from E. coli RY13 (R.N. Yoshimori)
Reagents Supplied:
NEBuffer 4 (10X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 10% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 0% | | NEBuffer 4: | | 100% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Blocked by some combinations of overlapping | |
More information about: Methylation Sensitivity |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
(+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours.
More information about: Extended Digests with Restriction Enzymes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Incubate at
37°C.
1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One 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.
Concentration:
20,000 units/ml and 100,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM KPO4
300 mM NaCl
10 mM 2-Mercaptoethanol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
0.15% Triton X-100
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent C
Notes
Usage notes:- EcoRI-HF™ has the same specificity as EcoRI (NEB #R0101), but it has been engineered for reduced star activity.
FAQs
- What does HF refer to following the name of the restriction enzyme?
- 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?
- When should I choose the High Fidelity (HF) version of the 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)?
- How does the level of star activity of EcoRI-HF compare to EcoRI?
- What is the difference between EcoRI-HF and EcoRI?
- What is the specific activity of EcoRI-HF?
- Is there any difference in the methylation sensitivity between EcoRI-HF and EcoRI?
- Is there a difference in cutting close to the ends between EcoRI-HF and EcoRI?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 20-fold overdigestion with EcoRI-HF™, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, > 95% can be recut with EcoRI-HF™.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 100 units of EcoRI-HF™ incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of EcoRI-HF™ with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (105 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of EcoRI-HF™ with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Blue/White Screening Assay:
An appropriate vector is digested at a unique site within the
lacZα gene with a 10-fold excess of enzyme.
The vector DNA is then ligated, transformed, and plated onto Xgal/IPTG/Amp plates.
Successful expression of β-galactosidase is a function of how intact its gene remains after cloning,
an intact gene gives rise to a blue colony, removal of even a single base gives rise to a white colony.
Enzyme preparations must produce fewer than 3% white colonies to be Blue/White certified.
Reagents Sold Separately
NEBuffer 4
Companion Products
EcoRI
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