Protocol for T5 Exonuclease (NEB #M0663)
- Set-up the following reaction on ice (Please note that the enzyme should be added last):
COMPONENTS 50 μl REACTION FINAL CONC. OR AMOUNT DNA up to 1 µg NEBuffer 4 (10X) 5 µl 1X T5 Exonuclease 1 µl 10 units Nuclease-free Water (NEB #B1500) to 50 µl
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- Incubate at 37°C for 30 minutes.
- Stop reaction with 6X Purple Gel Loading Dye (NEB #B7024 containing SDS) or add EDTA to at least 11 mM.
- To cleanup treated samples we recommend using a spin column (such as the Monarch® PCR & DNA Cleanup Kit, NEB #T1030), running the reaction on an agarose gel followed by extraction of the DNA (we recommend Monarch Gel Extraction Kit, NEB #T1020), or by 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.