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Quantify total DNA in small cell lysate...?

quantify DNA lysate

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#1 assembler01

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Posted 27 March 2013 - 08:21 AM

I have a bunch of 96-well TC plates (with mouse cells) that are snap frozen in 50uL of some kind of lysis buffer (100mM Tris pH7.4, 5mM EDTA pH8.0, 200mM NaCl, 1% triton x-100). I would like to quantify the DNA. We are attempting to find a way to quantify it with ethidium bromide, Hoescht or maybe some other fluorescent dye. If possible, we would also like to get out enough DNA to quantify the ratio of mitochondrial DNA to genomic DNA. What is the best way to proceed? I was thinking of doing a fluorescent assay for total DNA and then using what's left for PCR quantification of the ratio of mtDNA:gDNA. Unfortunately, I haven't seen it done much around here. Should I purify some or all of the lysate with the 'puregene' or other gDNA purification protocols? Can I just take a small sample of lysate directly to fluorescent quantification (either with DNA-specific dye, by spiking the sample for correction of quenching...should I add RNase then) and then another sample directly to qPCR?

Thanks.

#2 bigudukaz

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Posted 28 March 2013 - 12:57 AM

If you want to do it with as small sample as possible - you should go with qPCR. Primers (or even probes if you want) should be designed specifically for gDNA and other set for mtDNA. If you would quantify with gel or fluorescence - you will loose a lot of sample, probably all of it, and if something goes wrong you cant repeat experiment.
I guess you don't have a qPCR as this is not an option in your ideas described..





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