A Typical Exonuclease V Reaction (M0345)

Exonuclease V (RecBCD) efficiently degrades ssDNA and linear DNA, leaving supercoiled and nicked dsDNA intact.

1. Setup the following reaction:

 DNA up to 1 μg
 NEBuffer 4 (10X) 5 μl (1X)
 ATP (10 mM) 5 μl (1 mM)
 Exonuclease V 1 μl (10 units)
 Nuclease-free H2O  to 50 μl

2. Incubate at 37°C for 30 minutes.

3. To stop reaction add EDTA to 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: Estimate amount of DNA to be removed by OD260 or agarose gel electrophoresis. If  this is > 1 μg, scale up all reaction components proportionately.