Making sure your experiment goes right is a top priority because it saves time, money and prevents the overall frustration of the job. In many DNA extraction protocols, the use of proteinase K is an important step because of its ability to digest harmful nucleases, but how much to use, when to use it and for how long can sometimes be a mystery. In this article, we untangle 5 common proteinase K questions that relate closely to extraction methods. While we hope that this article serves as a helpful guide in your work, it is critical to do additional research to make sure your methods are perfectly matched to the type of work you’re doing.

To view a printable proteinase K digestion table, click here.

When is proteinase K used?

Proteinase K is used mostly in DNA and RNA extraction protocols. You’ll often find the proteinase K step within the lysis section of the protocol. For example, in the nucleic acid extraction protocol, proteinase K is added to cell lysate and then an incubation period follows to ensure a complete digestion.

To prevent potential digestion of your samples, proteinase K is inactivated after incubation. The common temperature for inactivation is 95°C.

Even in the typical mouse-tail protocol, proteinase K is regularly used to inhibit harmful nucleases. And the addition of proteinase K occurs during the digestion step. The use of EDTA is also suggested to help the inactivation of nucleases by inhibiting Mg2+ dependent nucleases.

Proteinase K Protocol and Digestion Guide

How do I know if digestion has happened?

Usually, the biggest tell that complete digestion has occurred is that you should see a clear lysed cell solution. If you are not seeing a clear solution after the initial digestion period, extend your incubation time.

Be very careful with this. If you are using a faster method for isolation, especially involving higher volumes of proteinase K, you’ll need to pay close attention during proteinase K digestion. A longer digestion may cause degradation of your DNA.

ProK Handbook download link

How long of an incubation time should I use?

The incubation period with proteinase K is going to depend primarily on the type of sample you’re working with. After doing quite a bit of research, here is the range of times we found for different cell and tissue samples. Please keep in mind that your experiment may have different requirements or variables that could greatly influence digestion times, and therefore we strongly encourage you to do your own research before carrying out your work.

Formalin-Fixed Paraffin-Embedded Tissues Digest for several hours to overnight.

*Note: Formalin-Fixed Paraffin-Embedded Tissues commonly appears in the abbreviated form, FFPE. This is a method for tissue preservation (another long-term tissue preservation method is with frozen tissue).

  • Bacteria – Digest with proteinase K between 1-3 hours. Digestion temperature may also influence how long your digestion should take.
  • Mammalian cells – There are several papers out there with a wide range of stated digestion times – as little as 1 hour and as long as twelve hours. This is partly due to experimental objectives and the type of cells used. Digestion temperature and proteinase K volumes also have some influence.

What temperature should I use for proteinase K digestion?

Digestion temperatures also vary with the type of sample you’re working with. Once again, we offer a guide on this, but strongly encourage you to do more research to optimize all conditions before proceeding with your experiment.

  • FFPE Tissue – Digestion temperatures 55-56°C. Most articles are fairly consistent with that temperature range.
  • Bacteria – Digestions are often carried out at 55°C. Some articles, however, did state a 37°C digestion temperature. Keep in mind the requirements of the type of sample you’re working with and other factors of your experiment.
  • Mammalian – Articles greatly varied in digestion temperatures. Shorter digestion periods usually correlated with higher temperatures (optimal proteinase K digestion temperatures for mammalian cells range between 50-65°C). Articles with digestions taking place for several hours to overnight usually suggested 37°C. Several other factors can impact the digestion temperature such as cell type (blood, buccal, etc.) and molecular weight.

How much proteinase K do I use?

The amount of proteinase K you need for successful digestion is going to depend on many factors: the protocol you’re using, the type of sample you’re working with, the conditions of your experiment, etc. Typically, 10-20 µl of proteinase K are used in experiments, with stock proteinase k stock concentrations usually around 20 mg/ml.

Something else to keep in mind is that some methods require a second digestion step (usually those involving tissue samples). Weaker, second digestions usually call for a lower volume and a different digestion period.

For more proteinase K tips, visit the product page for a list of related literature, or check out our articles on common questions about proteinase K and proteinase K activity.

Get your free Proteinase K Handbook with helpful articles, troubleshooting tips, protocols, product information and more all inside


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Proteinase K Protocol Digestion Guide

This guide shows the experimental conditions for digestion with proteinase K.