Hi,
I am using a technique in C2C12 muscle cells called DamID to tag DNA in close proximity to the nuclear envelope in undifferentiated myoblasts and differentiated myotubes. However, following sequencing of the tag-enriched material amplified by the final PCR step, 80% of the DNA sequences identified come directly from the mitochondrial genome. So it looks like the mtDNA is being tagged and because per copy it is more numerous than any genomic sequence, gets preferentially amplified.
I have tried removing the circular and supercoiled mtDNA by;
However, none of these have worked. Does anyone have any suggestions on easy ways to remove the mtDNA from the genomic DNA? The last thing I am thinking of trying is gel filtration?
Any help would be great!
Hi
I do not have better protocol to solve this problem, but want to ask do you try running agarose gel? Mitochondrial DNA should smaller than genomic DNA, so is it possible that use high % agarose gel to separate them?
This is just my guess.
1. The perinuclear region in some cells is anchored with nuclear membrane. Here the perinuclear region includes centrioles, MTOC, actin filaments, Golgi and parts of ER. Mitochondria are usually clustered in this perinuclear region by actin filaments/tubulins. In many cells you'll be able to see by immunofluorescence/GFP mitochondrial proteins that mitochondria are clustered at perinuclear region. Adding nocodazole or tubulin depolymerizing agents in your hypotonic lysis buffer will help... but you may not get 100% pure nuclear fraction to the level of PCR sensitivity.
2. This option is based on separating nuclear genome from mitochondrial genome based on size differences. Might need longer runs of gels with periodical buffer changes+ cooling.
Good Luck.
There is no way of subsequent removal of mitochondrial DNA from a total DNA preparation.
After a usual isolation DNA extraction procedure both nuclear and mitochondrial DNA is fragmented, consisting of linear fragments of 10 to 100 kb in size.
The only possibility is to isolate intact nuclei, remove any contaminating cell debris and mitochondria by differential centrifugation,
performing a light DNAse treatment, than extracting DNA from the lysed nuclei.
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