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Restriction Endonucleases >
Restriction Endonucleases >
NheI-HF™ |  | | | | NheI-HF™ | | | New | High-Fidelity Enzymes | |
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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:
Many restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed star activity. HF restriction endonucleases have been engineered to cleave with higher fidelity than the wild type enzyme, hence exhibiting less star activity.
Source:
An E. coli strain that carries the cloned and modified (E77A) NheI gene from Neisseria mucosa heidelbergensis (ATCC 25999)
Reagents Supplied:
NEBuffer 4
BSA
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 100% | | NEBuffer 2: | | 10% | | 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 |
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.13 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Supplemented with 100 μg/ml Bovine Serum Albumin
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 (HindIII digest) in 1 hour at 37°C in a total reaction volume of 50 μl.
Concentration:
20,000 units/ml
Unit Assay Substrate:
λ DNA (HindIII digest)
Storage Conditions:
10 mM Tris-HCl
250 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
0.15% Triton X-100
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent C
Notes
General notes:- Cleaves to leave a 5´ CTAG extension which can be efficiently ligated to DNA fragments generated by AvrII, SpeI or XbaI.
- Activity inhibited by salt concentrations > 100 mM.
Usage notes:- NheI-HF™ has the same specificity as NheI (NEB #R0131), but it has been engineered for reduced star activity.
FAQs
- What is the specific activity of NheI-HF™?
- What does HF refer to following the name of the restriction enzyme?
- 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?
- Is there any difference in the methylation sensitivity between NheI-HF and NheI?
- What is the difference between NheI-HF and NheI?
- When should I choose the HF version of the enzyme?
- When is star activity a concern?
- Is there a difference in cutting close to the ends between NheI-HF and NheI?
- What does it mean to be Time-Saver qualified?
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 100-fold overdigestion with NheI-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 NheI-HF™.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 100 units of NheI-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 200 units of NheI-HF™ with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 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 NheI-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.
Reagents Sold Separately
NEBuffer 4
BSA
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 6,387,681 B1
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