I mostly found different protocols which either pellet the mitochondria, lyse them and isolate the DNA or don't pellet the mitochondria and in this case, isolate the mtDNA from the bulk cell content obtained. In older protocols, a gradient of CsCl was used to separate the mtDNA. I guess the choice should go by the coming application of the mtDNA; in my case, I will further digest the mtDNA into dNucleosides.
I would do the same as Manuel Gutiérrez-Aguilar but I would treat the cells with zymoliase before then use a dounce or a potter instead of beads. Mitochondria are quite a fragile thing.
If money isn't an issue, try MitoSciences' Mitochondria Isolation Kit - you'll need a Dounce homogenizer for it to maintain mitochondrial integrity. If you don't have the homogenizer, there is a kit that includes it and is available from Abcam, but if you have the homogenizer, there's a kit without it.
Thanks a lot Manuel Gutiérrez-Aguilar and Rémy Saunier, I am following the zymolyase treatment, differential centrifugation to isolate the mitochondrial pellet and washing the mitochondrial pellet 3x with sorbitol, TE, mercaptoethanol buffer. Do you have any further clue on how can I get evidence that after all I don't have any remaining of nuclear DNA attached to my mitochondria?
You can try to perform a purification with sucrose gradient or histodenz. It will purify further your mitochondria but, with my little experience in science and mitochondria purification, you can not get rid of all the mito associated ER membrane (i.e. MAM). Hence you can not be sure to get rid of all the nuclear DNA. But you can try a little trick (I don't know if it works, though): mitochondrial DNA is "tiny" and circular and genomic DNA is "big" and linear, so that a miniprep like those used to purify plasmids from bacteria may purify a little bit more your mitochondrial DNA after the purification of mitochondria. But again, I'm not sure you can get rid of all the nuclear DNA even with all these steps.
You can verify the purity of your mitochondrial DNA preparation by running a real-time PCR assay with mtDNA and nuclear DNA primer sets. This will give you a relative enrichment of mtDNA/nDNA, and you can verify the purity of your preparation. You could also run your extracted DNA on an agarose gel, and gel extract the mtDNA band(s).
Easy, Just start by purifiying mitochondria from yeast as well as possible. A lot of purification procedures have been described. Then, extract you mtDNA....
Traditionally, all DNA was isolated together and then separated on a CsCl gradient. This normally results in quite clean mitochondrial DNA due to the different GC-content of mtDNA compared to gDNA. If you isolate mitochondria first, you will loose a lot of material and need larger cultures to start with.
@Mark : culture isn't a real problem for S. cerevisiae. Produce several liters (thus, several grams of yeasts) in liquid galactose rich media just takes O/N. The CsCl is another good option though.
@Remy: Yes, of course you are right. But as you said earlier, clean mitochondria are also difficult to isolate, hence the suggestion to do it the traditional way and isolate all DNA first, and then separate the nuclear from the mitochondrial on the CsCl gradient. The comparative qPCR suggested by Martin is a good one to check the final sample on purity.
From several methods I have tried, the only one that allows pure mtDNa is CsCl gradient. qPCR is great to chek. I am nearby, if you need any collaboration. Best,
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