Mitochondrial DNA Isolation Kit (ab65321)

Reviews (2)Q&A (48)References (13)

Overview

  • Product name

    Mitochondrial DNA Isolation Kit

  • Sample type

    Tissue, Adherent cells, Suspension cells

  • Assay type

    Direct

  • Assay time

    3h 00m

  • Product overview

    Mitochondrial DNA Isolation Kit ab65321 provides convenient tools for isolating mtDNA from a variety of cells and tissues in high yield and purity, without contaminations from genomic DNA. The purified mtDNA can be used for a variety of studies such as enzyme manipulations, Southern blotting, cloning, PCR analysis, and amplifications


    Please note: One kit is sufficient for 50 samples if Adherent cell or Suspension cells are used and for 25 samples if tissue is used.

    Mitochondrial DNA isolation protocol summary:
    - collect cells by centrifugation at 600 x g for 5 min and wash with PBS
    - centrifuge for 5 min and resuspend pellet in cytosol extraction buffer
    - incubate on ice for 10 min
    - homogenize in dounce tissue grinder
    - centrifuge for 10 min at 700 x g to pellet nuclei and intact cells
    - transfer supernatant and centrifuge at 10,000 g for 30 min
    - remove supernatant, resuspend pellet in cytosol extraction buffer and centrifuge at 10,000 g for 30 min
    - resuspend pellet (mitochondria) and lyse in mitochondrial lysis buffer for 10 min
    - add enzyme mix and incubate for 60 min
    - add ethanol and incubate for 10 min
    - centrifuge for 5 min and retain pellet (mitochondrial DNA)

  • Notes

    Mitochondria are semi-autonomous organelles which functions in aging process, apoptosis, anti-HIV drugs, and cancers. Mitochondrial DNA (mtDNA) has a very high mutation rate and the mutations on mtDNA appear to be related to certain diseases such as diabetes, Alzheimer's disease, and muscle disorders. Isolation and quantification of mtDNA are often required to study the relationships between the diseases and mtDNA.

Properties

Protocols

References

This product has been referenced in:

  • Stampar SN  et al. Linear Mitochondrial Genome in Anthozoa (Cnidaria): A Case Study in Ceriantharia. Sci Rep 9:6094 (2019). Read more (PubMed: 30988357) »
  • Drareni K  et al. GPS2 Deficiency Triggers Maladaptive White Adipose Tissue Expansion in Obesity via HIF1A Activation. Cell Rep 24:2957-2971.e6 (2018). Read more (PubMed: 30208320) »
See all 13 Publications for this product

Publishing research using ab65321? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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Question

I have a question regarding the mitochondrial DNA isolation kit ab65321. Is there any RNase added in any of the buffers (I guess it could be a component of the Enzyme Mix)? In case of added RNase, what type of RNase is it?

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Answer



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

This is RnaseA from Bovine pancreas.

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Question

One of our costumers is intending to use Mitochrondrial DNA Isolation Kit - ab65321 for Marine Invertebrate and Reptile specimen.

He knows that this kit hasn't been tested for that kind of samples yet. However, he has questions about the kit purity (quality of extraction):


1- What's the expected (acceptable) range of Mitochondrial DNA Isolation for ab65321 final product?

2- Is it possible to have any Nuclear DNA contamination in the final product given by ab65321?

3- Do you know what's the range of Nuclear DNA contamination expected for ab65321 final product?

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Answer

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.

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Mitochondrial DNA isolation

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
Mitochondrial DNA (mtDNA) isolation has been performed using mitochondrial DNA Isolation Kit (Abcam, ab65321) following the manufacturer’s instructions. MtDNA has been isolated by MRC5 fibloblasts in high yield and purity, without contaminations from genomic DNA. 2.22 x 106 cells have been used in each condition, the concentration of the purified mtDNA was then measured using NanoDrop 2000, and graph presentation has been performed using the GraphPad Prism Version 5.01a statistical package (GraphPad Software Inc., USA). A 1% agarose gel stained with EtBr was also run to confirm the correct size (~ 17kB) using a DNA ladder, the purity and the concentration of mtDNA, and it was imaged using Chemidoc MP Imaging System (Biorad, USA).
Personal feedback: Mitochondrial DNA isolation Kit can be safely used to isolate mtDNA in high yield and purity, by using a standard cell number in each condition, the effect of various treatments in the number of mitochondria can be easily measured.

Dr. Dimitra Kalamida

Verified customer

Submitted Nov 10 2016

Extraction of mtDNA from whole blood

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
This is a great kit to extract mitochondrial DNA. We just added an extra step of isolating Peripheral Blood Mononuclear Cells (PBMC) from whole blood and applied the abcam protocol (with minor changes, see attached protocol) to isolate mtDNA from PBMC.

Lina Abi Mosleh

Verified customer

Submitted Apr 20 2016

Question

Does the Enzyme Mix neutralize reactive oxygen species from mitochondria?

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Answer

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

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Question

My sample is whole DNA (genomic + mitochondrial). I'd like to potentially use this kit to isolate the mitochondrial DNA, but I see this kit is more designed for earlier on in the process when your sample is tissue or cells. Do you think the kit would work with my sample, and if so, do you have any protocol recommendations?

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Answer

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.

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Question

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?



Many thanks,


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Answer

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.

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Question

Inquiry: Hi there, I have a question about the Mitochondrial DNA Isolation Kit (ab65321). For my application I need the isolated mtDNA to remain in the circular form - do you know if the mtDNA isolated by the kit remains circular?

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Answer

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

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Question

Is it possible to get some information about the composition of the Cytosol Extraction Buffer and the Mitochondrial Lysis Buffer of the Mitochondrial DNA isolation kit (65321)? Thank you very much in advance.

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Answer

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.

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Question

I don't have a Dounce homogenizer. Which one do you recommend?

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Answer

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.

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