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To whom it may concern:
I am writing to you to enquire about the suitability of your kit for the extraction of mtDNA from frozen tissue for downstream epigenetic analysis; specifically, if you may be able to tell me if there are any products in your kit which may interfere with methylation studies. Also, your protocol for this kit suggests that 5-20ug of mtDNA will be returned from 107 cells, what would your expected return be on a 100mg tissue?
Asked on Apr 23 2014
Thank you for your enquiries regarding ab65321 Mitochondrial DNA Isolation Kit .
I have now obtained some further information from the laboratories.
As I thought, the Enzyme B composition is proprietary. However, I can confirm there should be no problem using the mtDNA for any DNA amplification/cloning application.
Ideally fresh tissue would be better for mtDNA isolation. But frozen tissue can be used. There should be no problem using the DNA for methylation studies. We do not use any chemicals that can damage or alter the DNA in any way during isolation. Some protocol modifications are suggested for tissue samples:
1. Use 3 fold more 1x cytosol Extraction buffer, so that the homogenized tissue will not be too sticky to remove the insoluble materials at low spin step.
2. Do step 11 without keeping on ice for 10 min, and then directly go to step 12, add 10 or 15 ul Enzyme B Mix, then incubate at 50 degree C overnight. Enzyme B mix will degrade all proteins and DNases.
3. With 50 million cells, 5-20ug mtDNA is obtained. However, please note that the yield is difficult to predict because it is different from tissue to tissue and prep to prep depending on how much mtDNA got digested by endonucleases. It also depends on the oxidative state of the cell. So if the yield is not enough for your purposes, simply scale everything up accordingly.
It might also help to review the following publication where other researchers have used this kit:
Gao X et al (2008) J. Biol. Chem.; 10.1074/jbc.M806235200.
There is a need to homogenize the tissue with a Dounce homogenizer.
The mtDNA can be run on a gel – 15-20 Kb circular DNA.
Hope this information is helpful. Please do not hesitate to contact us if you have any further questions or concerns.
Sam WasherAbcam Scientific Support
Answered on Apr 23 2014