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New England Biolabs Restriction Enzymes

Inside the Numbers

  • Highest buffer compatibility
  • Significantly reduced star activity
  • Time-saving
  • Economical

With over 40 years of offering restriction enzymes to the research community, New England Biolabs (NEB) has earned the reputation of being a leader in enzyme technologies. NEB offers scientists 276 restriction enzymes, the largest selection in the industry.

New England Biolabs
Restriction Enzymes

Highest buffer compatibility

> 205 restriction enzymes are 100% active in the single CutSmart™ buffer, making double digest reactions significantly easier to set up. Since many DNA modifying enzymes are also 100% active in CutSmart buffer, there is often no need for purification between reactions.

Small selection of the DNA modifying enzymes that show 100% activity in the CutSmart buffer

Enzyme Activity in CutSmart buffer Required Supplements
Alkaline Phosphatase (CIP) +++
T4 DNA polymerase +++
Shrimp Alkaline Phosphatase (rSAP) +++
T4 DNA Ligase +++ Requires ATP

+++ full functional activity ++ 50–100% functional activity
+ 0–50% functional activity

Visit for a complete overview of the DNA modifying enzymes.


Of the 20 biggest selling restriction enzymes from NEB, 19 enzymes are supplied with the CutSmart buffer and 1 is provided with its own buffer. The convenience of this unrivaled buffer compatibility means great savings to your budget and time.

Buffers supplied with the most commonly sold restriction enzymes
NEB Thermo Scientific Promega Roche
20 enzymes available with just 2 buffers 20 enzymes available with 9 different buffers 14 enzymes evailable with 5 different buffers 13 enzymes available with 4 different buffers

A table with the 20 biggest selling restriction enzymes can be found here

Example: Double digestion with BamHI and SalI

New England Biolabs

BamHI-HF® and SalI-HF® both exhibit 100% activity in the CutSmart buffer so each enzyme works at regular concentration to obtain 100% cleavage.

Enzyme Activity % in NEBuffers
1.1 2.1 3.1 CutSmart
BamHI-HF 25% 100% 100% 100%
SalI-HF 100% 25% 10% 100%


There is no buffer available in which both enzymes exhibit 100% activity. As these two enzymes are not compatible, Roche recommends performing a sequential digestion to get the job done.

Enzyme Activity % in Roche buffers
BamHI 100% 100% 75-100% 100% 25-50%
SalI 0-10% 25-50% 0-10% 10-25% 100%

Significantly reduced star activity

NEB has developed a line of High-Fidelity (HF®) restriction enzymes. These engineered enzymes have the same specificity as the native enzyme, with the added benefit of reduced star activity, rapid digestion (5-15 minutes) and 100% activity in CutSmart buffer.
Star activity can cause extra bands that confuse interpretation of results in genotyping and mutational analysis procedures. NEB HF enzymes are engineered to perform under a wide range of conditions, so whether digests are for 5-15 minutes or overnight, or using high amounts of enzyme, you can be confident of achieving the performance you need without star activity.
Example: Reduced Star activity of NEB EcoRI
NEB EcoRI-HF (#R3101) shows no star activity when used for 5 minutes or overnight. 50 μl reactions were set up using 1 μg of Lambda DNA, 1 μl of enzyme (NEB EcoRI-HF or Thermo Scientific Fermentas EcoRI) and recommended reaction buffer. Digests were incubated at 37°C.


Typically, a restriction digest means incubating 1 μl of enzyme with 1 μg of purified DNA in a final volume of 50 μl for 1 hour. For results in just 5-15 minutes, choose one of the 190 time-saver™ qualified enzymes from NEB.
Time-saver qualified enzymes digest 1 μg of substrate DNA in just 5-15 minutes using 1 μl of enzyme under recommended reaction conditions, and can also be used safely overnight. The fast results need no special formulation, or change in concentration – and there are no additional costs.
Example: Power and purity of time-saver qualified NEB HindIII-HF
NEB’s HindIII-HF (#R3104) is powerful enough to digest 1 μg of DNA in 5 minutes, but can also be used in overnight digests with no indication of nuclease contamination. Marker (M) is the 1 kb DNA Ladder (#N3232).


Price comparison for NEB’s biggest selling enzymes

Supplier Price 1.000 units** Difference*
NEB € 1.832,95
Thermo Scientific Fermentas Conventional € 1.805,10 -2%
FastDigest € 19.069,74 90%
Roche € 4.921,57 63%
Promega € 3.686,52 50%

* Average difference to NEB list price

** Price comparison (price of 1.000 units of the most-sold 20 enzymes) is based on similar size packs published in June 2014 by the suppliers on their website. Not all suppliers were able to provide all the 20 restriction enzymes, for these enzymes we used the average price.

Biggest selling New England Biolabs restriction enzymes

Cat # Enzyme Buffer
1. R3552S AgeI-HF
2. R0558S AscI
3. R3136S


4. R3535S BsaI-HF
5. R0176S DpnI
6. R0543S DpnII
7. R3101S EcoRI-HF
8. R3104S HindIII-HF
9. R3589S MfeI-HF
10. R3193S NcoI-HF
Cat # Enzyme Buffer
11. R0111S NdeI
12. R3131S NheI-HF
13. R3189S NotI-HF
14. R0547S PacI


16. R3138S SalI-HF
17. R3133S SpeI-HF
18. R0145S XbaI
19. R0146S XhoI
20. R0180S XmaI

= Cutsmart Buffer = Own unique NEBBuffer


High-Fidelity (HF) Restriction Enzymes:


Restriction enzymes


Recombinant enzymes


Enzymes exhibiting 100% activity in a single buffer


Time-Saver qualified enzymes


High-Fidelity Enzymes engineered

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