Mitochondrial DNA Isolation Kit (ab65321)

I can confirm there is RNase contained in the mitochondrial lysis buffer.

This is RnaseA from Bovine pancreas.

Thank you for contacting Abcam regarding ab65321.


Regarding this Mitochondrial DNA Isolation Kit:


1 - Expect to recover 5-20ug mtDNA

2- The most important step with this kit is to isolate a pure mitochondrial pellet and to separate the nuclei from the mitochondria. If this is not achieved then there will be some carry over of nuclear DNA. Hence, we would suggest to start with a defined amount of tissue and then lyse it in a fixed volume of the buffer by homogenisation. The most important step then is to optimise the number of passes using a Dounce homogenizer so as to not to attain overlysis. The optimal settings depend on the tissue type, so I am sorry that we cannot provide more specific recommendations on this occasion.

Also, the following centrifugation step to remove nuclei and intact cells (step 6, attached) has been optimised for 10 minutes at a G-force of 700. We would therefore recommend to set the spin speed of the centrifuge accordingly.


3 - When the protocol is followed as outlined, there should be no Nuclear DNA contamination.


I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

No, the Enzyme B Mix or any other component should not neutralize ROS species.

ab65321 is designed to isolate mitochondria first and then lyse the mitochondria to get the mtDNA. Thus, since you have a solution of DNA with genomic and mtDNA mixed in, then this kit is not designed to isolate the mtDNA specifically. One way the lab suggested is to run an agarose gel. Genomic DNA will be a smear and mtDNA would be between 16-20Kb and you can cut the gel piece out and purify DNA from this or do electroelution.

However, the best way to ensure mtDNA purity is to first isolate the mitochondria, then proceed with mtDNA extraction.

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.

I can confirm that the isolated mtDNA in the mitochondria isolated with this kit will be circular and will run at ˜15-20 kbs.

Vielen Dank für Ihre Geduld.

Leider ist diese Information ein Betriebsgeheimnis, doch das Labor ist gerne bereit, auf mehr spezielle Fragen einzugehen, wie zb. pH- Wert einzelner Reagenzien, oder Verwendung nach der Lyse.

Ich hoffe, dies hilft Ihnen weiter.Wir freuen uns auf ihre Antwort.

Thank you for contacting us.

We recommend the 2 mL version of this homogenizer:

https://ca.vwr.com/store/catalog/product.jsp?product_id=4830627

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.