A Typical Duplex DNase Reaction Protocol (NEB #M7635)

  1. Set up the following reaction on ice:
    NEBuffer r1.1 (10X)  5 µl (1X)
    Duplex DNase
    1 µl (2 units)
    Nuclease-free H2O
    to 50 µl
     *Substrate can be a mixture of ssDNA and dsDNA or a DNA/RNA hybrid.

  2. Gently mix the reaction by pipetting up and down and microfuge briefly.

  3. Incubate between 25-65°C for 5-30 minutes.

    *Note: Optimization of reaction conditions may be required based on experimental variables. We recommend varying reaction conditions (enzyme amount, time, temperature) for optimal results.

  4. To heat inactivate Duplex DNase, add DTT (1 mM final concentration) and incubate at 75°C for 10 minutes. If the sample contains RNA, we recommend adding EDTA (10 mM final concentration), along with DTT (1 mM final concentration) prior to heat inactivation as temperatures >65°C in the presence of divalent metals such as Mg2+ may degrade RNA.