Reduce Star Activity with High-Fidelity Restriction Enzymes

NEB has engineered HF® enzymes to eliminate star activity. Learn how, and what this means for your digests.


Rick Morgan:
New England's BioLab's High-Fidelity® or HF enzymes have been engineered to eliminate star activity and also to function in a single common buffer, the rCutSmart™ buffer. Which is especially helpful when doing double digests and the speed of the enzymes, and the lack of star activity allows you to create flexibility in terms of the time of reactions. They can be cut very quickly with Time-Saver™ or they can go overnight and still give you great fidelity. We created the HF enzymes by engineering specific functional residues, altering them and then selecting those enzymes that gave increased fidelity under often grossly unfavorable reaction conditions. For each of the HF enzymes we report the HF factor, which indicates how much more of the HF enzyme one can use in a reaction compared to the wild type, and still avoid having star activity or off site cutting.

The HF factor can be especially dramatic for some of the enzymes. For example, HindIII-HF® will give the same specific patterns, even if you use up to 2000 fold more enzyme compared to the wild type. One thing to remember with the HF enzymes is that they are engineered and they are different than the wild type enzymes. So the wild type conditions may not be the same as what you want to use for the HF enzymes. All of the HF enzymes from NEB are available for the same price per unit as the wild type enzymes. For more information you can go to our website for more information in general about the High-Fidelity® restriction enzymes

Note:  We are excited to announce that we are in the process of switching all reaction buffers to be BSA-free. Beginning April 2021, NEB will be switching our current BSA-containing reaction buffers (NEBuffer™ 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin (rAlbumin)-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). Find more details at


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