Protocol for cleavage with Tth Endonuclease IV (NEB #M0294)

1) Reaction set-up:

DNA Up to 10 pmol
ThermoPol Reaction Buffer (10X) 5 µl
Tth Endonuclease IV
1 µl (10 units)
H2O Up to 50 µl

2) Incubate at 65°C for 1 hour.


  1. * An AP site is created by treating 10 pmol of a 60-mer oligonucleotide duplex containing a single uracil residue with 1 unit of Uracil-DNA Glycosylase (UDG) for 2 minutes at 37°C.
  2. Place reaction in Thermocycler or overlay with mineral oil to reduce evaporation.
  3. Enzyme stability above 80°C is assured by adding ZnCl2 to a final concentration of 25 μM in the reaction.
  4. Not recommended in NEBuffers 1-4 and Thermopol II Buffer.