Protocol for T5 Exonuclease (M0363)

T5 Exonuclease will degrade ssDNA and linear and nicked dsDNA, leaving supercoiled dsDNA intact.

1. Set-up the reaction as follows:

DNA up to 1 µg
NEBuffer 4 (10X) 5 µl (1X)
T5 Exonuclease 1 µl (10 units)
Nuclease-free H2O (NEB #B1500) up to 50 µl

2. Incubate at 37°C for 30 minutes.

3. Stop reaction with 6X Purple Gel Loading Dye (NEB #B7024 containing SDS) or add EDTA to at least 11 mM. 

4. To cleanup treated samples we recommend using a column for cleanup (such as the Monarch® PCR & DNA Cleanup Kit, NEB #T1030), or running the reaction on an agarose gel and then extracting the DNA (we recommend Monarch Gel Extraction Kit, NEB #T1020), or 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.