Product Class: Restriction Endonuclease

NcoI-HF®
icon_cloned icon_recomb engineered icon_timesaver5 icon_CutSmart dil_B-grey icon_incTemp37 heat inactivation

Nco-I-cutsite_1

Product Introduction

High-Fidelity (HF®) restriction enzymes have the same specificity as native enzymes, but have been engineered for significantly reduced star activity and performance in a single buffer (CutSmart® Buffer).  All HF-restriction enzymes come with Gel Loading Dye, Purple (6X).  Enjoy the enhanced performance and added value of our engineered enzymes at the same price as the native enzyme:

  • Engineered for improved performance
  • 100% activity in CutSmart Buffer
  • Time-Saver™ qualified for digestion in 5-15 minutes
  • Reduced star activity
  • Supplied with 1 vial of Gel Loading Dye, Purple (6X)
Bioz Badge Exists : True

Product Information

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.

NEB extensively performs quality controls on all standard and high-fidelity (HF) restriction enzymes. Examples of nuclease contamination studies for some of our HF restriction enzymes are shown below.

Restriction Enzyme Competitor Study: Nuclease Contamination


EcoRI, NotI, and BamHI from multiple suppliers were tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3’overhang or 5’ overhang containing oligonucleotides. The percent degradation is determined by capillary electrophoresis and peak analysis. The resolution is at the single nucleotide level.

Product Source

An E. coli strain that carries the cloned and modified from Nocardia carolina (ATCC 19070)

Properties & Usage

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.

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)

Activity in NEBuffers

CutSmart® Buffer: 100%
NEBuffer™ 1.1: 50%
NEBuffer™ 2.1: 100%
NEBuffer™ 3.1: 10%

Diluent Compatibility

Storage Temperature

-20°C

Storage Conditions

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

Heat Inactivation

80°C for 20 min

Methylation Sensitivity

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

Product Notes

  1. NcoI-HF® has the same specificity as NcoI (NEB #R0193), but it has been engineered for reduced star activity. 
  2. Not sensitive to CpG, dcm, or dam methylation.

Protocols, Manuals & Usage

Protocols

  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes
  3. Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
  4. Restriction Digest Protocol
  5. Protocol for Digestion Prior to droplet digital PCR (ddPCR)

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. Is there a difference in cutting close to the ends between NcoI-HF and NcoI?
  2. What is the difference between NcoI-HF and NcoI?
  3. How does the level of star activity of NcoI-HF compare to NcoI?
  4. When should I choose the HF version of the enzyme?
  5. When is star activity a concern?
  6. Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
  7. When should I choose the High Fidelity (HF®) version of the enzyme?
  8. Can the change in buffer preference of the HF enzyme be advantageous?
  9. Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended?
  10. What does it mean to be Time-Saver™ qualified?
  11. How is the improvement in fidelity of HF restriction endonucleases quantitated?
  12. What does HF® refer to following the name of a restriction enzyme?
  13. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  14. 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?
  15. 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?
  16. Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?
  17. Why is my Restriction Enzyme not cutting DNA?
  18. Why do I see a DNA smear on an agarose gel after a restriction digest?
  19. Why do I see additional DNA bands on my gel after a restriction digest?
  20. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?
  21. Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)?
  22. Is this enzyme sensitive to dam, dcm or mammalian CpG methylation?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

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]

Certificate Of Analysis

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

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.