Product Class: Restriction Endonuclease

NotI-HF®

This enzyme has transitioned to an improved new buffer system. Visit NEBCutSmart.com for further details.
 
The new and current Double Digest Finder and current Activity/Performance Chart for the CutSmart buffer system are available. The previous version of the Double Digest Finder, as well as the previous Version of Activity/Performance Chart that use the former buffer system, are still available for your convenience.

High Fidelity Enzymes

Product Update: A free vial of Gel Loading Dye, Purple (6x) is now included with all HF restriction enzymes. Please note there may be a slight delay for freezers and international products as inventory is switched over.
cloned at neb recombinant engineered timesaver 5min unique buffer incubation temp heat inactivation cpg
Not-I-cutsite_1
Catalog #SizeConcentration
R3189S500 units20,000 units/ml
R3189L2,500 units20,000 units/ml
R3189M2,500 units100,000 units/ml

Description

  
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 Source

An E. coli strain that carries the cloned and modified (K150A) NotI gene from Nocardia otitidis-caviarum (ATCC 14630)

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
CutSmart™ Buffer-2010X
Gel Loading Dye, Purple (6X)256X

Properties and Usage

Unit Definition

One unit is defined as the amount of enzyme required to digest 1 μg of pBC4 DNA in 1 hour at 37°C in a total reaction volume of 50 μl.

Reaction Conditions

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

Diluent Compatibility

Storage Temperature

-20°C

Storage Conditions

10 mM Tris-HCl
50 mM NaCl
1 mM DTT
0.1 mM EDTA
200 μg/ml BSA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 20 min

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Blocked

Heat Inactivated

Yes

Quality Control

Quality Control Assays

The 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.

Supporting Documents

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Material Safety Datasheets

The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Notes

  1. NotI-HF® has the same specificity of NotI (NEB #R0189), but it has been engineered for reduced star activity. 
  2. Supercoiled plasmids may require up to 5-fold more NotI-HF for complete digestion on linear DNAs.
  3. Blocked by CpG methylation.
  1. What does HF refer to following the name of a restriction enzyme?
  2. When is star activity a concern?
  3. Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
  4. When should I choose the High Fidelity (HF) version of the enzyme?
  5. Can the change in buffer preference of the HF enzyme be advantageous?
  6. Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended?
  7. What does it mean to be Time-Saver™ qualified?
  8. How is the improvement in fidelity of HF restriction endonucleases quantitated?
  9. What is the Fidelity Index (FI)?
  10. How does the level of star activity of NotI-HF compare to NotI?
  11. What is the difference between NotI-HF and NotI?
  12. What is the specific activity of NotI-HF?
  13. Is there any difference in the methylation sensitivity between NotI-HF and NotI?
  14. Is there a difference in cutting close to the ends between NotI-HF and NotI?
  15. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  16. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  17. How can I access the old NEBuffer Activity Chart?
  18. How can I access the old Double Digest Finder?
  19. I tested your restriction enzyme on the substrate DNA recommended by NEB, and it appears to be active, however it does not digest my DNA. What could be the reason?