High Fidelity Enzymes

Product Update: A free vial of Gel Loading Dye, Purple (6x)  is now included with all HF restriction enzymes.

cloned at neb recombinant engineered timesaver 5min icon_CutSmart dil_B-grey incubation temp heat inactivation dam
Catalog #SizeConcentrationPriceQtyAdd to Cart
R3160S3,000 units20,000 units/ml$58.00Add to Cart
R3160L15,000 units20,000 units/ml$234.00Add to Cart
High-Fidelity (HF®) Restriction Endonucleases,
Restriction Endonuclease Products,
Restriction Endonucleases: B
Restriction Enzyme Digestion


Product Source

An E. coli strain that carries the cloned and modified BclI gene from Bacillus caldolyticus (A. Atkinson).

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 λ DNA (dam) 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: 100%
NEBuffer 3.1: 10%
CutSmart® Buffer: 100%

Diluent Compatibility

Storage Temperature


Storage Conditions

10 mM Tris-HCl
300 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: Blocked
dcm methylation: Not Sensitive
CpG Methylation: Not Sensitive


  1. BclI-HF has the same specificity as BclI (NEB #R0160), but it has been engineered for reduced star activity.
  2. Cleaves to leave a 5´ GATC extension which can be efficiently ligated to DNA fragments generated by BamHI, BglII, MboI, Sau3AI and BstYI.
  3. This enzyme is blocked by dam methylation. More information can be found at Dam-Dcm and CpG Methylation.
  4. Star activity may result from a glycerol concentration of >5%


  1. What does HF® refer to following the name of a restriction enzyme?
  2. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?    
  3. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  4. How can I access the old NEBuffer™ Activity Chart?
  5. Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?
  6. Why is my Restriction Enzyme not cutting DNA?
  7. Why do I see additional DNA bands on my gel after a restriction digest?
  8. Why do I see a DNA smear on an agarose gel after a restriction digest?
  9. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

Tech Tips

This enzyme will not cut plasmids isolated from most E.coli because it is blocked by dam methylation. For enzyme cleavage to occur, plasmids must be transformed and isolated from a dam-, dcm- strain of E.coli, such as NEB’s dam-/dcm- Competent E.coli (NEB #C2925).


  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes
  3. Time-Saver Protocol for Restriction Enzyme Digests

Selection Charts

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

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. Further information regarding NEB product quality can be found here.
  • 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, DNA):
    The DNA is tested in a reaction under standard reaction conditions. After incubation for 16 hours there is no detectable degradation of the DNA as determined by agarose gel electrophoresis.

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]

Safety Data Sheet

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