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DNA extraction from dissociated embryos


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#1 Johannes

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Posted 19 June 2014 - 05:59 PM

Good morning everyone.

 

I have been tasked to run sex determining PCRs on a batch of bovine embryos. Unfortunately the straws containing the embryos have been mailed all over the country a couple of times and in between seem to have been stored inadequately. Therefore some of the embryos have dissociated and I can not find and discern the actual embryo from whatever else is floating around in the medium. My PCR works fine if I can find an embryo, put it in a new cup, lyse it with Proteinase K and add the rest of the PCR reaction to the same tube. 

 

The problem is that I do not want to examine every embryo straw under the microscope in the hope that there is still an intact one in there, because of the fear that the dissociated cells might stick to the dish and I loose everything.

 

Due to the low amount of cells I would also prefer to not run a separate DNA extraction.

 

I was thinking about emptying the content of the straw into a 0.2 ml PCR tube (it's about 150 micro litres) and concentrate the cells by centrifugation, then cautiously remove as much liquid as possible and add Proteinase K.

 

I looked around and found suggestions about centrifuging mammalian cells that range from 200 - 2000 g. Does anyone have experienced anything similar before and maybe has a couple of suggestions on how I should continue?

 

Kind regards,

 

Johannes



#2 bob1

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Posted 19 June 2014 - 07:34 PM

Depending on what they have been frozen in, there could be quite a few PCR inhibitors floating around in a tube, so it may be best to dilute the straws in PBS or a buffered saline of some sort, then spin down the cells.  Ice crystal formation rupturing the cells will be the biggest issue you have in getting the cells out intact - thaw them fast to 37 in a waterbath.

 

Spinning down of cells works well at pretty much any speed less than 500 RCF, higher can cause the cells to rupture and leak contents (which you obviously don't want)



#3 Johannes

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Posted 22 June 2014 - 05:23 PM

thanks bob,

I tried two of my samples. Spun them for 30 minutes at 500 RCF, removed most of the supernatant and added the proteinase K solution. After digestion I added the rest of the PCR mastermix and the reaction worked nicely. So whatever they have been frozen in did not seem to interfere with my reaction. 






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