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:
|COMPONENTS||50 μl REACTION|
|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.