I am facing the problem of genomic DNA contamination in my mitochondrial DNA isolation from insect tissue. I want to include a DNAse treatment of my whole mitochondrial pellet before proceeding to cell lysis and DNA extraction. Can anyone suggest me the buffer composition to prevent my mitochondria from undergoing osmotic stress and at the same time be effective for DNAse activity as PBS is ineffective and DNAse buffer obtained with DNAse is also not preventing the mito DNA from degrading.
Have you tried isolating the mitochondrial DNA by treating the tissue as if you were isolating plasmid from Bacteria? A standard alkaline lysis ( NaOH/SDS, K-acetate etc. ) style isolation should trap genomic DNA while the circular mitochondrial DNA would partition like a plasmid.
Daniel I am using the alkaline lysis method already. I am doing the process in two stages. first i am isolating the mitochondria using sucrose gradient method and then proceeding to alkaline lysis of them to extract the DNA. I think there may some nuclear dna remaining attached to the mitos which is not getting removed in further prcoessing. Are you suggesting that i take whole tissue directly to alkaline lysis?
I think you could.Alternatively, any "next step" that allows you to separate by size ( I recall that human mitcohondrial DNA is about 16 or 17 kbp- should remove contaminating genomic DNA- a low percentage agarose gel for example, or if you have lots of tissue and only a few different samples, a CsCl gradient.
thanks daniel but the thing is i am very short of whole insect tissue. I cant risk using whole insect. I am getting results with my method which i chose after a lot of search. I am sequencing the dna i get but the genomic dna is resulting in generation of unwanted contigs. I am to map most of the mito genome but i just wish to improve the quality of the reads by removing any gDNA reads. Thats why i thought of giving DNAse treatment.
I am yet to find proper conditions...but still thanks for suggesting the option.
One more option you might consider if you want to try a nuclease is to use lambda Exonuclease. If you are careful isolating the DNA the mitochondrial will be circular and not be a substrate for this exonuclease ( one of the few that won't act on gaps or nicks). NEB sells this enzyme. You would follow-up with a single stranded exonuclease like exo I.
Hello!! I'd like to ask one thing. I have isolated Chloroplast from leaves with percoll gradient. Chloroplast, observed under a microscope were very beautiful. After I extracted DNA with a DNA extraction kit and amplified with chloroplast genes. I think that in my chloroplast DNA there was nucleus contamination... how can I obtain a Ch DNA without contaminations???
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