Protocol for Exonuclease VIII, truncated (M0545)
Exonuclease VIII, truncated efficiently degrades linear dsDNA from 5’ to 3’ direction, 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)|
|Exonuclease VIII (truncated)||1 µl (10 units)|
|Nuclease-free H2O (NEB #B1500)||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:
- Column clean up (we recommend the Monarch® PCR & DNA Cleanup Kit, NEB #T1030) or
- Running the reaction on an agarose gel, and then extracting the DNA (we recommend the 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.