Protocol for Exonuclease III (M0206)

Exonuclease III efficiently degrades nicked and linear dsDNA (with blunt or 5' overhangs) from 3' to 5' direction, leaving supercoiled dsDNA intact.*

1. Set-up the reaction as follows:

 DNA  up to 5 μg
 NEBuffer 1 (10x)
 5 μl (1X)
 Exonuclease III
 0.5 μl (50 units)
 Nuclease-free H2O
 up to 50 μl


2. Incubate at 37°C for 30 minutes.

3. Stop reaction by adding EDTA to at least 11 mM.

4. Heat inactivation 70°C for 30 minutes.

5. To clean up treated samples, we recommend using one of the following steps:

a. Column clean up (we recommend the Monarch® PCR & DNA Cleanup Kit, NEB #T1030), or

b. Running the reaction on an agarose gel, and then extracting the DNA (we recommend the Monarch Gel Extraction Kit, NEB #T1020), or

c. Performing a phenol/chloroform extraction followed by ethanol precipitation.

*Note: For more precise results or partial digestions, we recommend titration of the enzyme to the intended substrate.