To inhibit DNase activity you can use several methods

1) Inhibit the enzyme activity

Low temperature reduces the optimal temperature conditions for enzymes activity. Keeping DNA on ice helps with reducing enzyme activity

Chemical Inhibitors of DNases include:

2-mercaptoethanol (Laskowski 1971)

2-nitro-5-thiocyanobenzoic acid (Moore 1981)

Actin (Mannherz et al. 2008, and Lazarides and Lindberg 1974)

Alfatoxin B2a, G2, G2a, and M1 (non-competitive) (Schabort 1970)

Ca2+ (Moore 1981)

EGTA (Moore 1981) and EDTA (Junowicz and Spencer 1973)

Sodium dodecyl sulfate (Liao 1975a)

Calf spleen inhibitor protein (Laskowski 1971)

Carbodiimide and cholesterol sulfate (Iwamori 2000)

Iodoacetate (Moore 1981)

2) reduce enzyme number by capturing proteins from the nuclear extract or cytosolic extract. You can precipitate out proteins and clear most Dnases

3) use double distilled purified water when storing and or creating buffers for isolating DNA

4) never use pipettes used for other tasks such as protein isolation. Always clean pipettes carefully and DNAse treat before using for PCR or DNA extraction

5) Always use sterlized / autoclaved equipment and tubes

6) DNA is best used FRESH, and stored at -20 C preferrably or - 80 C for long term storage. Other methods include lyophilized, or paper dried storage for long term storage to prevent the aqueous environment needed for enzyme activity

To inhibit DNase activity you can use several methods

1) Inhibit the enzyme activity

Low temperature reduces the optimal temperature conditions for enzymes activity. Keeping DNA on ice helps with reducing enzyme activity

Chemical Inhibitors of DNases include:

2-mercaptoethanol (Laskowski 1971)

2-nitro-5-thiocyanobenzoic acid (Moore 1981)

Actin (Mannherz et al. 2008, and Lazarides and Lindberg 1974)

Alfatoxin B2a, G2, G2a, and M1 (non-competitive) (Schabort 1970)

Ca2+ (Moore 1981)

EGTA (Moore 1981) and EDTA (Junowicz and Spencer 1973)

Sodium dodecyl sulfate (Liao 1975a)

Calf spleen inhibitor protein (Laskowski 1971)

Carbodiimide and cholesterol sulfate (Iwamori 2000)

Iodoacetate (Moore 1981)

2) reduce enzyme number by capturing proteins from the nuclear extract or cytosolic extract. You can precipitate out proteins and clear most Dnases

3) use double distilled purified water when storing and or creating buffers for isolating DNA

4) never use pipettes used for other tasks such as protein isolation. Always clean pipettes carefully and DNAse treat before using for PCR or DNA extraction

5) Always use sterlized / autoclaved equipment and tubes

6) DNA is best used FRESH, and stored at -20 C preferrably or - 80 C for long term storage. Other methods include lyophilized, or paper dried storage for long term storage to prevent the aqueous environment needed for enzyme activity

I wish to express my full appreciation for your quick response!

I would to ask you one more thing.

My DNAseI assay protocol uses 0.5 M EDTA solution (10X) to inhibit the enzyme. However, the inhibition does not work very well (especially for zero time) and putting it in ice the inhibitory effect improves but not enough.

Then, I was wondering if the calcium carried by EDTA Ca Na2 salt (stock reagent available in my lab.), may reduce the inhibitory capacity of EDTA?

What you think about that?

Hi Alex,

EDTA works by immediately chelating Mg2+ ions that are required for DNaseI activity, so don't use a Ca-Na salt of EDTA (EDTA also chelates Ca2+, so you're right, your solution will dramatically reduce the inhibitory capacity of EDTA).

To irreversibly inactivate DNaseI, heat your sample for 10-15 min at 65degC (or 1 h at 55degC if you have a sensitive sample) AFTER adding EDTA to the sample.

This procedure works well for me, I've even stored DNA afterwards for 6 months at 4degC and it was still OK, so there was no residual activity left.