NEBcutter® V3.0 - Find restriction enzymes to remove a specific DNA sequence

This video tutorial explains how to find restriction enzymes to remove a specific DNA sequence.


NEBcutter is a free tool that is used for restriction enzyme analysis. There are many different ways to use this tool. In this video, you will learn how to find restriction enzymes to remove a specific DNA sequence. 

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. Next, set your submission preferences.  This is where custom oligos are defined. The default options work for most situations. You can also designate if your DNA molecule is linear or circular. If your sequence is circular, please select "circular”.  Please see the help icon for more information about the preference options. Press “submit”.

After submitting your sequence, NEBcutter displays a map of your sequence. By default, it displays enzymes with unique sites within your sequence. If you would like to search for enzymes to remove a specific Open Reading Frame (ORF) within your DNA sequence, click the “flanking sites” option on the Graphical View page. Unique restriction sites flanking each of the identified ORFs will be displayed.

If you would like to generate a list of enzymes to remove a specific region of your DNA sequence, click the “List” tab under “Display”. On this page, enter the region of DNA to be excised at the top of the page. The display shows RE sites flanking your selected region. The distance flanking your region of interest can be adjusted from 200bp (default) to a larger or smaller area. Select “Closest sites with compatible ends” if the cleaved ends need to be ligated. If there are no unique restriction sites within the defined area, the region of interest can be removed by inverse PCR. Methylation dependent RE can then be used to remove the starting template assuming it was made in a methylation+  host strain.

Please note that the Graphical View and Flanking Sites views are independent with respect to the region selected.
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