Therefore, we would think it's unlikely that RNA in the sample would be causing problems. It's possible there are interfering factors in the sample. Rather than bother with normalization, which could be difficult if their dna concentration is very low, They could simply dilute their sample before running the PCR to see if there's any interference. I hope these suggestions are also helpful. After incubation at 55C for 2 hours the proteinase K would most likley be entirely degraded by itself.
If there is any active proteinase K remaining, the ethanol precipitation may denature the protein. Then, when performing the PCR, the customer only uses a small amount of the sample thus diluting the proteinase K further. If the customer wants to be certain, they could simply incubate their sample at 95C now to deactivate any potentially active protease , and then run the PCR reaction again.
More than likely the customer is actually losing their DNA in the ethanol precipitation step. If the DNA concentration is very low, then it will not precipitate in ethanol, and they will lose it when they remove the supernatant. Please keep us informed if your next tests are more positive or if you continue having mixed results.
October 22, at am. Anna, I have seen it affect things down the line, when it was not inactivated. It can affect some things further down the line on some protocols. It can depend on the extraction methodology. The 90 degrees will also help denature for various types of protocols. A bunch of things I don't see from your steps that I first think about that might affect your PCR runs. Your extraction method of precipitation can carry over other items. Is there a PAGE step or other steps of extraction being done?
Normalization to make sure you are not overloading. October 21, at pm. I still normally spin the tissue after 2hours of 55C incubation with proteinase K buffer for 20 mins at room temperature, then I put the supernatant in fresh tubes and add cold ethanol, precipitating DNA overnight AT c. Hello Jeremy, and David, thank you both for your quick responses. We have been researching different ways of breaking down hair in the lab at school for the purpose of metals identification.
This is for nutrition and or foreign substances testing. In my studies I came across a research study on Proteinase K and they touch on the subject of it breaking down human hair, finger nails, and the 1st layer of human skin. However for hair they still ended up using a pestel and mortar to crush the hairs. I was looking for a way to do this specifically with a solution.
Applying heat would be acceptable. Once again thanks to both of you for your responses. October 19, at pm. Casey, I guess you can find out yourself. In theory yes, but it would take a very longtime. If you use heat 37 degrees Celcius , there is a better chance of it. The concentration of your Proteinase K will also affect the reaction rate. Jeremy, I am not sure I can give you more information considering the parameters that you are doing. I am not familiar with all those cell lines.
I would use heat inactivation. I grabbed this tidbit from the Promega Website. Proteinase K may not be completely inactivated by EGTA, as this enzyme retains partial activity in the absence of calcium 7. October 18, at am. Jeremy McGuire. October 6, at am. I want to use proteinase K on the conditioned media before treating the prostate cancer cells. So I would like to know what is the best concentration of proteinase K to use and the best way to inactivate it heat or Pefablock Thanks for any advice.
October 3, at pm. Jeremy, I am not sure what you are asking? Are you trying to find the correct concentration levels of Proteinase K?
I am thinking Proteinase K would kill cells if it is not inactivated. High heat has been shown to inactivate Proteinase K. What cell line are you using? Why are you neutralizing with Pefablock?
For the Pefablock. Do a pH reading of just the media and add Pefablock to see if it affects pH. October 3, at am. I wanted to use proteinase K to treat conditioned media. I have come across various protocols in the literature. Does anyone have experience in using proteinase K for this purpose?
I am trying to find the best way to determine if the PK is working. I think the Pefablock lowered the PH of my conditioned media. Any advice will be appreciated. September 20, at pm. I'm freeze-lysing whole blood sample with the goal of RNA recovery. I've been adding proteinase k after thawing the blood, but it would be ideal to freeze the blood with proteinase k, then thaw and have immediate nuclease digestion, any thoughts on freezing proteinase k?
Would it still be functional? September 8, at am. Beta mercaptoethanol is a denaturing agent that affects protein structure. It does not serve as the same function as Proteinase K.
Numerous disulfide bonds make ribonucleases very stable enzymes, so 2-mercaptoethanol is used to reduce these disulfide bonds and irreversibly denature the proteins. This prevents them from digesting the RNA during its extraction procedure. To view our range of antigens and antibodies for immunoassay development please visit our partner site meridianlifescience.
Language Worldwide. Proteinase K. Product Highlights High activity - broad-spectrum serine protease Active under denaturing conditions - useful for a variety of applications Stable - at high temperatures Molecular biology grade - useful for general digestion of proteins Proteinase K is also available as a stabilized stock solution.
What is the optimal pH for proteinase K? Proteinase K is active in a pH between 7. What is the primary sequence for proteinase K? What is Proteinase K?
References: Breyer, J. Then came college, and the revelation that the adults in my past were right all along. But since math feels less tangible, I fell for biology and have found pure happiness behind my desk at GoldBio, learning, writing and loving everything science.
Leave Feedback. Related Articles. Making sure your experiment goes right is a top priority because it saves time, money and prevents A lot of questions pop up when it comes to using proteinase K, such as how to inactivate proteinase Press down.
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DNA isolation from 1—10ml blood samples. Liquid-handler compatible. We use these cookies to ensure our site functions securely and properly; they are necessary for our services to function and cannot be switched off in our systems. They are usually only set in response to actions made by you which amount to a request for services, such as logging in, using a shopping cart or filling in forms. You can set your browser to block or alert you about these cookies, but some parts of our services will not work without them.
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