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DNA extraction from non-viable PBMCs

concentration

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#1 Rashmi

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Posted 24 December 2012 - 07:18 PM




Hey everyone,

I am trying to extract genomic DNA from 1 million peripheral blood mononuclear cells. Cells were pelleted and were frozen without any kind of freezing media. So, I am guessing that most of them are non-viable.
I used a kit to extract DNA and I got around 1.5 to 2ug total DNA (10ng/ul in 200ul elution volume).

I was just wondering if this yield is ok or if I am losing a lot of DNA? The protocol says that total yield from viable PBMCs should be around 6-8ug. The extracted DNA has a 260/280 ratio of 1.87 and 260/230 ratio of 0.97.
I want to use the extraction DNA for a taqman open array.

I will really appreciate if you guys can share your views.


Thanks,
Rashmi



#2 Curtis

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Posted 25 December 2012 - 09:25 PM

the concentration is low. but I think you can continue to the next experiment with it.

#3 Myworkismyplay

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Posted 26 December 2012 - 07:47 PM

I assume the expected yield is from 1 million cells? Viability is the wrong term to use here. As soon as that cell pellet was frozen all of the cells died. You are referring to quantity and quality of genomic DNA from frozen, lysed cells.

The sample seems much too dilute to check integrity and your concentration is most likely way off judging by how low it is and your A260:A230 ratio. Even if you are using NanoDrop, you are really below the accurate threshold for that spec. A better & more accurate method is the Qubit (DNA kit) to tell you how much DNA is really there, or if your A260 I just the residual side peak of the absorbance spectrum dropping past 260nm on the way down from 230nm, where your contaminants & buffer/salt residual material would absorb.

Most likely what you have in that 200 uL is impure nucleic acid, of a very low concentration.

If you are determined to use this particular sample, I suggest a clean-up ethanol precipitation to first bring your volume down, as well as increase the concentration so your spec reading can be more reliable. This will also remove some of the impurities you see at the A230. If done properly, you should recover >95% of your sample.

I would not carelessly toss this "DNA" onto an array and assume the gDNA is of good enough quantity, and especially not of good enough quality to have meaningful, reliable results for your research project.

If you can get a fresh PBMC sample that's what I would start with-- to get quality sample, before wasting money and time with a bad sample. Do the kit extraction protocol according to procedure and see if your sample improves. It should be easy to get good gDNA from PBMCs.

Good luck!





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