Product Class: Restriction Endonuclease

cloned at NEB recombinant engineered time saver CutSmart_long dil_A 37 No


Product Introduction

Not sensitive to dam, dcm or mammalian CpG methylation.

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
Catalog # Size Concentration
R3136S 10,000 units 20,000 units/ml
R3136T 10,000 units 100,000 units/ml
R3136L 50,000 units 20,000 units/ml
R3136M 50,000 units 100,000 units/ml

Product Information


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 BamHI gene from Bacillus amyloliquefaciens H (ATCC 49763)

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
Product Categories:
High-Fidelity (HF®) Restriction Endonucleases Products,
Restriction Endonucleases B,
time-saver-qualified-restriction-enzymes Products
Restriction Enzyme Digestion

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to digest 1 ug 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

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

Diluent Compatibility

Storage Conditions

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

Heat Inactivation


Methylation Sensitivity

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

Product Notes

  1. BamHI-HF® has the same specificity as BamHI (NEB #R0136), but it was engineered for reduced star activity.
  2. The increased specificity for the BamHI-HF® cut site has increased binding of the enzyme to the DNA.  Under enzyme excess conditions the enzyme can remain attached to the DNA during gel electrophoresis. To disrupt binding, please use the supplied 6X Purple Gel Loading Dye (NEB #B7024S) or add SDS to a final concentration of 0.1% - 0.5%.  Alternatively to  purify the DNA we recommend Monarch PCR & DNA Cleanup kit, NEB #T1030) before electrophoresis.
  3. For enzymes that cannot be heat-inactivated, we recommend using a column for cleanup (such as the Monarch® PCR & DNA Cleanup Kit), or running the reaction on an agarose gel and then extracting the DNA (we recommend Monarch Gel Extraction Kit), or performing a phenol/chloroform extraction.
  4. Not sensitive to CpG, dcm, or dam methylation.

Protocols, Manuals & Usage


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

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting


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


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.


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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.