Product Class: Restriction Endonuclease

icon_cloned icon_recomb icon_timesaver5 icon_NEBU dil_C-grey icon_incTemp37 icon_HI_65 icon_cpg
Item# Description List Price Web Price Qty
R0101L EcoRI - 50,000 units $349.00
R0101M EcoRI (100,000 units/ml) - 50,000 units $349.00
R0101S EcoRI - 10,000 units $86.00
R0101T EcoRI (100,000 units/ml) - 10,000 units $86.00
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at


Product Introduction

Having supplied restriction enzymes to the research community for over 40 years, NEB has earned the reputation of being the leader in enzyme technologies. Working continuously to be worthy of that distinction, NEB strives to develop enzymes of the highest purity and unparalleled quality.

Bioz Badge Exists : True

Product Information


EcoRI has a High Fidelity version EcoRI-HF® (NEB #R3101).

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 minutes 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.

For details on NEB’s quality controls for restriction endonucleases, visit our Restriction Enzyme Quality page.

Product Source

An E. coli strain that carries the cloned EcoRI gene from E. coli RY13 (R.N. Yoshimori).

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 NEBuffer™ EcoRI
Incubate at 37°C

1X NEBuffer™ EcoRI
100 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
0.025% Triton® X-100
(pH 7.5 @ 25°C)

Activity in NEBuffers

CutSmart® Buffer: 50%
NEBuffer™ 1.1: 25%
NEBuffer™ 2.1: 100%
NEBuffer™ 3.1: 50%

Diluent Compatibility

Storage Temperature


Storage Conditions

10 mM KPO4
300 mM NaCl
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
1 mM DTT
0.15% Triton® X-100
pH 7.0 @ 25°C

Heat Inactivation

65°C for 20 min

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Not Sensitive
CpG Methylation: Blocked by Some Combinations of Overlapping

Product Notes

  1. May exhibit star activity in NEBuffer 2.1 or CutSmart Buffer.
  2. This enzyme has shown to have lower activity on some supercoiled plasmids, with more than 1 unit required to digest 1 μg plasmid DNA. For complete digestion of 1 μg of plasmid DNA please follow our recommended digestion protocol..
  3. Blocked by some combinations of overlapping CpG methylation.

Protocols, Manuals & Usage


  1. Optimizing Restriction Endonuclease Reactions
  2. Double Digest Protocol with Standard Restriction Enzymes
  3. Restriction Digest Protocol

Usage & Guidelines

  • Activity at 37°C for Restriction Enzymes with Alternate Incubation Temperatures
  • Activity of Restriction Enzymes in PCR Buffers
  • Cleavage Close to the End of DNA Fragments
  • Cleavage of Supercoiled DNA
  • Digestion of Agarose-Embedded DNA: Info for Specific Enzymes
  • Double Digests
  • Effects of CpG Methylation on Restriction Enzyme Cleavage
  • Heat Inactivation
  • NEBuffer Activity/Performance Chart with Restriction Enzymes
  • Optimizing Restriction Endonuclease Reactions
  • Reduced Star Activities of HF® Enzymes
  • Restriction Endonucleases - Survival in a Reaction
  • Restriction Enzyme Diluent Buffer Compatibility
  • Restriction Enzyme Tips
  • Single Letter Codes
  • Site Preferences
  • Star Activity
  • Traditional Cloning Quick Guide

Tools & Resources

Selection Charts

  • Alphabetized List of Recognition Specificities
  • Buffer and Diluent Formulation Table
  • Compatible Cohesive Ends and Generation of New Restriction Sites
  • Cross Index of Recognition Sequences
  • Dam-Dcm and CpG Methylation
  • Frequencies of Restriction Sites
  • Isoschizomers
  • Recleavable Filled-in 5' Overhangs
  • Why Choose Recombinant Enzymes?

Web Tools

  • Competitor Cross-Reference Tool
  • DNA Sequences and Maps Tool

FAQs & Troubleshooting


  1. What is Star Activity and how can it be avoided?
  2. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
  3. Why is my Restriction Enzyme not cutting DNA?
  4. Why do I see a DNA smear on an agarose gel after a restriction digest?
  5. Why do I see additional DNA bands on my gel after a restriction digest?
  6. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?
  7. Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)?
  8. Is this enzyme sensitive to dam, dcm or mammalian CpG methylation?


  • Restriction Enzyme Troubleshooting Guide

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.