NEBcutter® V3.0 - Determine the restriction enzymes that cut your DNA

This video tutorial describes how you can use NEBcutter to find out which restriction enzymes can be used to cut your DNA sequence a specific number of times.


NEBcutter is a free tool that is used for restriction enzyme analysis.  There are many different ways to use the tool. In this video, you will learn how to find out which restriction enzymes can be used to cut your DNA sequence a specific number of times.

First, enter your DNA sequence into NEBcutter. You can either paste, upload or choose from one of the pre-loaded sequences available in the tabs. You can also select a sequence from a previous project. You can designate if your DNA molecule is linear or circular. If your sequence is circular, please select "circular”. Click submit. 

On the default “Graphical View” page, you can select “1 cutters”, “2 cutters”, “3 cutters” or “List 0 cutters”. For a full list of REs with recognition sites within the DNA molecule, select “Custom Digest”. Select enzymes of interest and then click “Digest” to visualize where the enzymes cut on the DNA molecule. The digestion information can be displayed as a “graphical view”, “enzyme list”, “fragment” list, or “gel”. Gel view will display digestion patterns for unmethylated and/or methylated DNA substrate.
The Enzyme List view also shows a full list of RE sites in the sequence by selecting n-cutters and setting “min” to 1; you can leave “max” blank. This feature provides a list of all the restriction enzymes that will cut the DNA, number of recognition sites, recognition sequence of enzyme, and enzyme activity in all four NEBuffers.

Note: 1, 2, and 3 cutters are particularly useful when trying to linearize plasmid DNA or determine which enzyme to use for RFLP analysis.
Note 2: 0 cutters are useful for engineering unique restriction sites into DNA molecules. First ensure that the site does not occur in the plasmid construct. Then introduce the restriction site flanking the region of DNA to be removed.

Visit to get started!

Related Videos

  • StandardREProtocol_video_thumb
    Standard Protocol for Restriction Enzyme Digests
  • EnzymeNotCutting_video_thumb
    Why is My Restriction Enzyme Not Cutting DNA?
  • DoubleDigestProtocol_video_thumb
    NEB® Restriction Enzyme Double Digest Protocol
Visit NEB’s Video Library
Loading Spinner