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In part II. "10.To the 10 ml fraction that contains the most...

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Minglin Lin Oct 5, 2016
protocol Protocol: Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells
In part II. "10.To the 10 ml fraction that contains the most crypts, add 10 ml Basal Media containing 15 U/ml DNase I (add 30 μl to 10 ml Basal Media) and then filter through a 100 μm filter into a BSA (1%) coated 50 ml Falcon tube". What is the exact role of DNase I here? Is it essential? Read more Read less more less
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Minglin Lin Answered Oct 6, 2016

SIU

Ok, get it. Thank you very much!
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Kevin Patrick O’Rourke Author Answered Oct 6, 2016

Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program

The DNase is used to digest the free floating DNA that arises as a result of cell lysis during the incubation and trituration steps. If you don't add the DNAse, you will notice that your fraction will be "soupy" and will not easily pass through the filter, and so after you clog one filter you will have to replace it with a new one and repeat. So, while it is not essential, it will increase your yield and decrease the number of filters required to finish filtering your fraction. I am not sure why other protocols use Type IX Collagenase, if they report success with this reagent and you have it readily in your lab then I don't see why you can't give it a try. Historically, we've had success with Type IV collagenase and so that's what we choose to use. Good luck with your protocol and let me know if you have any other questions! -Kevin O'Rourke
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