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.