Protocol for T5 Exonuclease (NEB #M0663)

1. 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

2. Gently mix the reaction by pipetting up and down and microfuge briefly.
   
   
3. Incubate at 37°C for 30 minutes.
   
   
4. Stop reaction with 6X Purple Gel Loading Dye (NEB #B7024 containing SDS) or add EDTA to at least 11 mM. 
   
   
5. 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.