Restriction Digestion

This protocol is validated

The validation document for this protocol is available here


This protocol allows you to cleave DNA fragments or plasmids using restriction enzymes. All you need to provide is the DNA you would like to digest, and select an enzyme from our library. You can also decide to run a gel for validation at the end of your digestion!

Sample requirements



Select at least one aliquot of DNA to digest. Each aliquot will be digested by the enzyme you select in a separate reaction.

Required parameters


Total Reaction Volume

Specify the total volume of the reaction you would like. This includes DNA, buffer and enzyme.

DNA Volume

Specify the volume of DNA you would like included in your reaction. Up to 1µg of DNA per reaction is suggested.


Select from our list of enzymes in our commercial inventory. Please email if you don't see the enzyme you are looking for.

Enzyme volume

Specify the amount of enzyme you would like included in each reaction. In general, we recommend 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest.

Digestion Conditions

Please specify the digestion conditions that you would like for your reaction. Most enzymes perform optimally at 37C for a 1 hour digestion. Please see documentation on you enzyme of choice by visiting's NEB's Enzyme Finder.

Include uncut control?

Checking this will prepare an extra digestion reaction using each DNA sample with no enzyme for an uncut control. 5µl of each uncut control reaction will be run on a 0.8% agarose gel.

Run Gel?

Check to visualize the result of your digestion reactions on a 0.8% agarose gel. Checking this will use 5µl of each digestion reaction to run on the gel. This option is chosen automatically is you choose to include an uncut control.

Ladder size

If you choose to run a validation gel. please select which ladder you would like run beside your samples on your gel.

High range ladder: 500bp, 1000bp, 2000bp, 4000bp, 10,000bp

Low range ladder: 100bp, 500bp, 1000bp, 2000bp

Protocol Outputs

This protocol outputs a new aliquot of the ligated sample which is stored in your inventory at -20°C . Any aliquots used in the making of the dilutions will be returned to the storage condition from which they came.

If you choose to include an uncut control, or run a gel, an annotated image of your gel is returned on the results tab of your run.

Recommended Guidelines

To optimize your reaction, please look up NEB's recommendations by finding your enzyme on NEB's Enzyme Finder. .

Ensure you are aware of the dead volume in the containers you are using for sample input (if you are using a diluent from your inventory). Dead volumes can be found in the containers section here. Failure to take into account dead volumes can result in insufficient volume being pipetted.

Further reading

Check out the forum if you have more questions.

For further advice on your protocol please ask questions to the community at