Troubleshooting NEBExpress^® Salt Active Nuclease (NEB #M0764)

DNA/RNA are not degraded as expected.

Reaction conditions not optimal

NEBExpress Salt Active Nuclease optimal activity is at pH ≥8.5, NaCl ≥500 mM. Outside of these conditions, add more enzyme or incubate longer. NEBExpress Salt Active Nuclease required at least 1 mM MgCl₂.

Inhibitor present in the reaction buffer

Chelator like EDTA >1 mM, reducing agent like DTT>1 mM or TCEP>0.1 mM will inhibit the enzyme activity.  Imidazole >20 mM, Triton X100 and Tween 20 >2% and phosphate buffers are not recommended.

Not enough enzyme added

Test various U of enzyme per mL of reaction.

Incubation time too short

Increase incubation time or temperature of reaction if possible. NEBExpress Salt Active Nuclease is active up to 50°C.

My sample is still viscous after following the procedure.

Not enough enzyme added or incubation time too short

Test various U of enzyme per mL of reaction or increased incubation time. We recommend an incubation temperature around 20-25°C for a quick and efficient procedure if possible.

Viscosity is not due to nucleic acids

One option is to dilute the viscous sample in the appropriate buffer and mix gently.

I think NEBExpress Salt Active Nuclease is losing its activity.

Inappropriate storage conditions, presence of high concentrations of chelators, detergents, or reducing agents.

NEBExpress Salt Active Nuclease is a very stable enzyme. It is still active after several days at room temperature or at 37°C and after multiple freeze thaws when kept in its storage buffer.

Still, inappropriate storage conditions or addition of chelating, reducing agents, proteases, glycerol >5%, detergents can damage or inactivate the enzyme. Change of buffer (low pH, phosphate buffers) will lower the enzyme activity.