A Typical Duplex DNase Reaction Protocol (NEB #M7635)
- Set up the following reaction on ice:
COMPONENTS 50 µl REACTION Substrate
x
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.
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- 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.
- 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.