Protocol for Exonuclease VIII, truncated (M0545)

Exonuclease VIII, truncated efficiently degrades linear dsDNA from 5’ to 3’ direction, leaving supercoiled dsDNA intact.

1. Set-up the reaction as follows:

DNA up to 1 µg
NEBuffer 4 (10X) 5 µl (1X)
Exonuclease VIII (truncated) 1 µl (10 units)
Nuclease-free H2O (NEB #B1500) 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:

  1. Column clean up (we recommend the Monarch® PCR & DNA Cleanup Kit, NEB #T1030) or
  2. Running the reaction on an agarose gel, and then extracting the DNA (we recommend the Monarch Gel Extraction Kit, NEB #T1020), or
  3. 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.