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:
| COMPONENTS | 50 μl 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.