Extraction of insect mitochondrial DNA - (Oct/09/2012 )

I am facing little trouble with confirmation of removal of nuclear DNA from my mtDNA preparation. I am using the alkaline lysis method to islate mitos and then DNA from them using CTAB method. How ever I am not sure if there are any nucler DNA traces in it or the DNA i isolate is infact the mtDNA. Can sombdy suggest a good way of identifiaction of insect mtDNA?

Maybe you could use PCR. Check one gDNA encoded gene which is present in multiple copies, e.g. 18S rRNA, as well as a mtDNA gene, e.g. mito 12S rRNA, from the same sample (in different reaction tubes). If you have enough DNA you also could check with Southern Blot. Maybe you can use DNase before you lyse your mitochondria?

Don't forget the positive controls to show your PCR worked properly! It is always difficult to show the "absence" of something because if you can not see it this doesnt mean necessarily it is not present.

thats a good option to use genomic DNA specific primers but I have many mtDNA specific primers used in earlier studies but with they sometime give result with mtDNA while sometime with gDNA. a few primers were specific only for mtDNA.. I was searching for some method which reduces the chance of any genomic DNA contamination like DNAse treatment....