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:
|COMPONENTS||50 μl REACTION
|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.