How might I deactivate a nuclease treatment?

Dear Esteemed Colleague,

Thank you for reaching out with your inquiry on how to deactivate nuclease treatments effectively. Nuclease enzymes, such as DNase or RNase, are crucial in molecular biology protocols for removing unwanted nucleic acids. However, their activity must be precisely controlled and terminated once the desired digestion has been achieved to prevent damage to valuable DNA or RNA samples. Below, I outline several approaches to deactivate nuclease treatments efficiently:

Heat Inactivation: Many nuclease enzymes can be inactivated by heating. The specific temperature and duration depend on the type of nuclease used. For example, DNase I can often be inactivated by heating at 65°C to 75°C for 10 to 20 minutes. It is important to consult the enzyme's technical datasheet for the exact conditions, as overheating can also degrade sensitive biomolecules.

EDTA Addition: Nucleases such as DNase and RNase require divalent cations (e.g., Mg2+ and Ca2+) for activity. Adding EDTA (Ethylenediaminetetraacetic acid) chelates these metal ions, effectively inhibiting nuclease activity. The concentration of EDTA needed can vary, but a final concentration of 2-5 mM is commonly used. Note that EDTA can interfere with downstream applications that require divalent cations, so its use must be considered carefully.

Protease Treatment: Nucleases are proteins; thus, treatment with proteolytic enzymes (e.g., Proteinase K) can degrade them and halt their activity. Following protease treatment, it may be necessary to inactivate the protease itself, often through heat inactivation or specific protease inhibitors.

pH Adjustment: The activity of nucleases can be highly pH-dependent. Adjusting the pH of the reaction mixture to extreme values (either acidic or basic), which are outside the optimal range for nuclease activity, can effectively denature and inactivate these enzymes. Care must be taken to ensure that the pH adjustment does not harm the nucleic acids or interfere with subsequent experimental steps.

Specific Inhibitors: Certain nucleases can be inactivated by specific chemical inhibitors. These inhibitors bind to the nuclease and block its activity without the need for heat or chelating agents. Availability and applicability depend on the specific nuclease in question.

Dialysis or Column Purification: To remove nucleases from a sample, dialysis against a nuclease-free buffer or column purification steps can be employed. These methods do not inactivate the enzyme per se but can effectively separate the nuclease from the nucleic acid sample.

When selecting a method for deactivating nuclease treatments, consider the nature of the nuclease, the sensitivity of your nucleic acid samples to the conditions used for inactivation, and the requirements of your downstream applications. It may also be prudent to perform a control experiment to assess the efficiency of nuclease inactivation and the integrity of the nucleic acids post-treatment.

I hope this information assists you in your molecular biology endeavors and contributes to the success of your experiments.

Best regards,

This protocol list might provide further insights to address this issue.

Misha Soskine

heat and EDTA will help with DNAse I (I guess phages are resistant to heat)

I'm not sure if RNAse A can be easily inactivated

Steingrimur Stefansson

Hi April, you could try proteinase K or trypsin treatment to degrade proteins.

Alexander I. Taylor

Ambion sell an inactivation/removal resin in one of their TURBO DNase kits that works well:

https://www.lifetechnologies.com/order/catalog/product/AM1907?ICID=search-product

Hsin-Yeh Hsieh

Heating at 65C for 5 min will kill it but not degrade the DNA.

Jai Ghosh

Instead of all the complicated heat treatment I would recommend to change the pH of the solution and simply freeze it in -20C.

Naveen Chaudhary

RNase even can survive in autoclave heat,but still traditional method is heat at 80 C and then keep in -20 C for few days ,then check its purity using nanodrop,sometimes initially gives good DNA reading but later on DNA degrades.

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