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Thread: Quantifying Cell Lysis or the Presence of DNA

  1. #1 Quantifying Cell Lysis or the Presence of DNA 
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    Hi,

    I am a Mechanical Engineering PhD student working on a microfluidics device to be used to perform DNA analysis. Part of this project includes lysing cells in a sample. We are considering several different methods of lysis that might work well on our small scale. In this project we would want to lyse bacteria and mammalian cells.

    The problem I am having is with determining whether or not the cells have been lysed, and quantifying what percent has been lysed. We have a Vanguard 1486 FL fluorescent microscope (http://veegee.thomasnet.com/item/flu...486fl?&seo=110) in which at 100x objective magnification, I can clearly see the red blood cells which we are attempting to lyse. However it is hard to tell even if the cells have been lysed.

    What might be some easy and relatively cost effective ways of determining cell lysis? Would there be a way to use some particular dye or fluorescence? In the end our goal is to extract the DNA from the cells to be analyzed in the device. So at this point I would like some way to quantify either if the cells have been lysed, or if DNA is present in the sample.

    Any advice you could give would be greatly appreciated.

    Thanks,
    Kevan


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  3. #2  
    Forum Cosmic Wizard i_feel_tiredsleepy's Avatar
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    You're trying to get DNA out of red blood cells...

    Um well spectrophotometers can be used to quantify DNA, use a known amount of DNA as a standard.


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    Forum Cosmic Wizard paralith's Avatar
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    Hi Kevan,

    You may want to visit this forum: http://www.protocol-online.org/forums/index.php
    It specializes in laboratory protocols, and you'll probably find more people familiar with the specifics of your situation there.
    Man can will nothing unless he has first understood that he must count on no one but himself; that he is alone, abandoned on earth in the midst of his infinite responsibilities, without help, with no other aim than the one he sets himself, with no other destiny than the one he forges for himself on this earth.
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  5. #4  
    Forum Cosmic Wizard i_feel_tiredsleepy's Avatar
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    I'd recommend using something like mouse hepatocytes or something if you're trying to get DNA, there isn't any DNA in red blood cells.
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  6. #5  
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    Ditto Paralith's link.

    Are you affiliated with a university. A chappy at my institution uses microfluidics in combination with pyrosequencing, I have some familiarity with the protocols and you could pm me.

    Short of more details, the obvious way to know if your protocol is working is to start with known controls of both cell types, dilution series, and QPCR on the resulting lysate to see if you are having the expected copy number in your lysate.
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  7. #6  
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    Quote Originally Posted by i_feel_tiredsleepy
    You're trying to get DNA out of red blood cells...

    Um well spectrophotometers can be used to quantify DNA, use a known amount of DNA as a standard.
    Thanks for the replies! I am currently just using a natural unseparated blood sample for the tests. I spoke of red blood cells because I just wanted to see if I have been able to lyse larger size cells before moving down in size to the bacteria, etc.

    Thanks,
    Kevan
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  8. #7 Re: Quantifying Cell Lysis or the Presence of DNA 
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    Quote Originally Posted by KevSC1
    Hi,

    I am a Mechanical Engineering PhD student working on a microfluidics device to be used to perform DNA analysis. Part of this project includes lysing cells in a sample. We are considering several different methods of lysis that might work well on our small scale. In this project we would want to lyse bacteria and mammalian cells.

    The problem I am having is with determining whether or not the cells have been lysed, and quantifying what percent has been lysed. We have a Vanguard 1486 FL fluorescent microscope (http://veegee.thomasnet.com/item/flu...486fl?&seo=110) in which at 100x objective magnification, I can clearly see the red blood cells which we are attempting to lyse. However it is hard to tell even if the cells have been lysed.

    What might be some easy and relatively cost effective ways of determining cell lysis? Would there be a way to use some particular dye or fluorescence? In the end our goal is to extract the DNA from the cells to be analyzed in the device. So at this point I would like some way to quantify either if the cells have been lysed, or if DNA is present in the sample.

    Any advice you could give would be greatly appreciated.

    Thanks,
    Kevan
    If DNA is what you are interested in I suggest that you first isolate the DNA. Just lysis of the cells will give you also all the cell crap, RNA, proteins, and other shit.

    DNA purification.
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  9. #8 Re: Quantifying Cell Lysis or the Presence of DNA 
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    Quote Originally Posted by spuriousmonkey
    If DNA is what you are interested in I suggest that you first isolate the DNA. Just lysis of the cells will give you also all the cell crap, RNA, proteins, and other shit.

    DNA purification.
    Well yes, this is going to be done in the actual device, after the cells are lysed, the DNA will be separated out of the sample. However I am just trying to focus on the cell lysis at the moment, I would just like something simple to see how well the various forms of cell lysis are working.

    Thanks,
    Kevan
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  10. #9 Re: Quantifying Cell Lysis or the Presence of DNA 
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    Quote Originally Posted by KevSC1
    Hi,

    The problem I am having is with determining whether or not the cells have been lysed, and quantifying what percent has been lysed. We have a Vanguard 1486 FL fluorescent microscope (http://veegee.thomasnet.com/item/flu...486fl?&seo=110) in which at 100x objective magnification, I can clearly see the red blood cells which we are attempting to lyse. However it is hard to tell even if the cells have been lysed.
    Have you tried DAPI or some other dye specific to nucleic acid? That might not help with RBC (I am not certain what DAPI-stained RBC look like) but if you look at other largish cells a DNA stain might give you some idea if the guts have spilled out.
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  11. #10  
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    Quote Originally Posted by i_feel_tiredsleepy
    You're trying to get DNA out of red blood cells...
    Better to try and get red blood cells out of a stone.
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