Protocol for T7 Exonuclease (M0263)

T7 Exonuclease efficiently degrades nicked and linear dsDNA (with blunt or 3' overhangs) from 5' to 3' direction, leaving supercoiled dsDNA inctact.*

1. Set-up the reaction as follows:

Components 50 μl REACTION
 DNA up to 1 μg
 NEBuffer 4 (10x)
 5 μl (1X)
 T7 Exonuclease
 1 μl (10 units)
 Nuclease-free H2O
 up to 50 μl

2. Incubate at 25°C for 30 minutes.

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

4. 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.