NEBcutter® V3.0 – How to determine methylation effects on restriction digests

This video tutorial explains how to use NEBcutter V3.0 to determine if your DNA of interest will be impacted by methylation during a restriction digest.


How to determine methylation effects on restriction digests using NEBcutter.

Some restriction enzymes can be impaired by overlapping DNA methylation as indicated by these product icons found on the product page. Let’s look at two ways you can use NEBcutter to determine whether DNA methylation will impact restriction sites in your DNA 

In the example, we have a plasmid that we would like to digest with the restriction enzyme XbaI. XbaI can be impaired by overlapping dam methylation. First select the plasmid pAd2-AvrII. Select “Circular”. Then click “Submit”. Read any warnings that appear and click “continue”.

There are 2 ways to view if a restriction enzyme site is impaired by methylation. We will first use the Enzyme list feature. Click “Enzyme List” and scroll to “XbaI”. 

The methylation sensitivity column indicates what methylation pattern may disrupt DNA digestion. Clicking on the arrow expands the list of XbaI sites. Here you can see that two XbaI sites are blocked by overlapping methylation. You can click on a specific cut position to get detailed information about the overlapping methylation. 

A second way to view the impact of methylation on a restriction digest is to use the Custom Digest feature. Again, we are interested in XbaI specifically. Using a Custom Digest will allow us to examine only XbaI sites. Click “Custom Digest” from the left menu. Select XbaI from the enzyme list by scrolling or typing in XbaI in the search box. Click “Digest”. The resulting custom digest plasmid map shows the XbaI restriction sites. Restriction Enzyme sites that are impacted by methylation are indicated with the following symbols. More information is available by clicking the ‘help’ icon.  The two XbaI sites labeled with the hash (#) symbol will be impaired by overlapping bacterial methylation if grown in a Dam+ host strain.. 

Please note that clicking on a restriction site will display detailed site information. 

NEBcutter can be found online at 


Related Videos

  • EnzymeNotCutting_video_thumb
    Why is My Restriction Enzyme Not Cutting DNA?
  • nebcloner_dd_demo_thumb
    Double Digestion with NEBcloner
  • StandardREProtocol_video_thumb
    Standard Protocol for Restriction Enzyme Digests
Visit NEB’s Video Library
Loading Spinner