For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome

We use cookies to make our site as useful as possible.

Our Cookie Policy explains how you can opt-out of the cookies we use.

If you continue without changing your cookie settings, we'll assume you’re happy with this.

Continue Continue

United States
Your country/region is currently set to:

If incorrect, please enter your country/region into the box below, to view site information related to your country/region.

Call (888) 77-ABCAM (22226) or contact us
Need help? Contact us

  • My account
  • Sign out
Sign in or Register with us

Welcome

Sign in or

Don't have an account?

Register with us
My basket
Quick order
Abcam homepage

  • Research Products
    By product type
    Primary antibodies
    Secondary antibodies
    ELISA and Matched Antibody Pair Kits
    Cell and tissue imaging tools
    Cellular and biochemical assays
    Proteins and Peptides
    By product type
    Proteomics tools
    Agonists, activators, antagonists and inhibitors
    Cell lines and Lysates
    Multiplex miRNA assays
    Multiplex Assays
    By research area
    Cancer
    Cardiovascular
    Cell Biology
    Epigenetics
    Metabolism
    Developmental Biology
    By research area
    Immunology
    Microbiology
    Neuroscience
    Signal Transduction
    Stem Cells
  • Diagnostic & Therapeutic Solutions
    Custom solutions & partnerships

    Custom antibody development and commercial partnerships to advance your diagnostic and therapeutic discovery.

    Create custom solutions with us

    Partner with us

  • Support
    Support hub

    Access advice and support for any research roadblock

    View support hub

    Protocols

    Your experiments laid out step by step

    View protocols

  • Events
    • Conference calendar
    • Cancer
    • Cardiovascular
    • Epigenetics & Nuclear signaling
    • Immunology
    • Neuroscience
    • Stem cells
    • Tradeshows
    • Scientific webinars
    Keep up to date with the latest events

    Full event breakdown with abstracts, speakers, registration and more

    View global event calendar

  • Pathways
    Cell signalling pathways

    View all pathways

    View all interactive pathways

Supporting our customers and employees during the COVID-19 pandemic. Read more

  1. Link

    mitochondrial-dna-isolation-kit-ab65321.pdf

  1. Send me a copy of this email
    I agree to the terms and conditions.
Kits/ Lysates/ Other Tools and Reagents DNA
Share by email

Mitochondrial DNA Isolation Kit (ab65321)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (2)Q&A (48)References (17)

Product price, shipping and contact information

Currently unavailable

Sorry, we can't display this right now.

Please contact us to place your order, or try again later.

 

Loading size & price…

 

Shipping and order information

Abpromise

Guaranteed product quality, expert customer support.

Find out more.

Key features and details

  • Assay type: Direct
  • Assay time: 3 hr
  • Sample type: Adherent cells, Suspension cells, Tissue

You may also be interested in

Chromogen
NBT / BCIP Solution - Ready to Use Alkaline Phosphatase chromogen (ab7468)

View more associated products

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

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 1 kit
    5X Cytosol Extraction Buffer 1 x 20ml
    Enzyme Mix (lyophilized) Red 1 vial
    Mitochondrial Lysis Buffer Purple 1 x 1.8ml
    TE Buffer 1 x 1.5ml
  • Research areas

    • Kits/ Lysates/ Other
    • Tools and Reagents
    • DNA
    • Kits/ Lysates/ Other
    • Kits
    • Molecular Biology Kits
    • Cell Fractionation Kits
    • Kits/ Lysates/ Other
    • Kits
    • Molecular Biology Kits
    • DNA Isolation Kits
  • Relevance

    Mitochondria are semiautonomous organelles which function in the 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.

Associated products

    Protocols

    • Protocol Booklet

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (17)

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

    ab65321 has been referenced in 17 publications.

    • Casciano JC  et al. MYC regulates fatty acid metabolism through a multigenic program in claudin-low triple negative breast cancer. Br J Cancer 122:868-884 (2020). PubMed: 31942031
    • Cai Y  et al. Elimination of senescent cells by ß-galactosidase-targeted prodrug attenuates inflammation and restores physical function in aged mice. Cell Res N/A:N/A (2020). PubMed: 32341413
    • Stampar SN  et al. Linear Mitochondrial Genome in Anthozoa (Cnidaria): A Case Study in Ceriantharia. Sci Rep 9:6094 (2019). PubMed: 30988357
    • Kaiser S  et al. Neuroprotection after Hemorrhagic Stroke Depends on Cerebral Heme Oxygenase-1. Antioxidants (Basel) 8:N/A (2019). PubMed: 31635102
    • Wang Y  et al. Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK. Cell Rep 29:1511-1523.e5 (2019). PubMed: 31693892
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-10 of 50 Abreviews or Q&A

    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?

    Read More

    Abcam community

    Verified customer

    Asked on Nov 19 2015

    Answer



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

    This is RnaseA from Bovine pancreas.

    Read More

    Sam Washer

    Abcam Scientific Support

    Answered on Nov 19 2015

    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?

    Read More

    Abcam community

    Verified customer

    Asked on May 02 2012

    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.

    Read More

    Abcam Scientific Support

    Answered on May 02 2012

    Mitochondrial DNA isolation

    Excellent Excellent 5/5 (Ease of Use)
    Abreviews
    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
    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?

    Read More

    Abcam community

    Verified customer

    Asked on Mar 23 2015

    Answer

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

    Read More

    Abcam Scientific Support

    Answered on Mar 23 2015

    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?

    Read More

    Abcam community

    Verified customer

    Asked on Aug 07 2014

    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.

    Read More

    Kevin Hanson

    Abcam Scientific Support

    Answered on Aug 07 2014

    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,


    Read More

    Abcam community

    Verified customer

    Asked on Apr 23 2014

    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.

    Read More

    Sam Washer

    Abcam Scientific Support

    Answered on Apr 23 2014

    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?

    Read More

    Abcam community

    Verified customer

    Asked on Apr 18 2013

    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.

    Read More

    Abcam Scientific Support

    Answered on Apr 18 2013

    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.

    Read More

    Abcam community

    Verified customer

    Asked on Jan 28 2013

    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.

    Read More

    Abcam Scientific Support

    Answered on Jan 28 2013

    Question

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

    Read More

    Abcam community

    Verified customer

    Asked on Dec 18 2012

    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.

    Read More

    Abcam Scientific Support

    Answered on Dec 18 2012

    1-10 of 50 Abreviews or Q&A

    •  Previous
    • 1
    • 2
    • 3
    • 4
    • 5
    • Next 

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

    Get resources and offers direct to your inbox Sign up
    A-Z by research area
    • Cancer
    • Cardiovascular
    • Cell biology
    • Developmental biology
    • Epigenetics & Nuclear signaling
    • Immunology
    • Metabolism
    • Microbiology
    • Neuroscience
    • Signal transduction
    • Stem cells
    A-Z by product type
    • Primary antibodies
    • Secondary antibodies
    • Biochemicals
    • Isotype controls
    • Flow cytometry multi-color selector
    • Kits
    • Loading controls
    • Lysates
    • Peptides
    • Proteins
    • Slides
    • Tags and cell markers
    • Tools & Reagents
    Help & support
    • Support
    • Make an Inquiry
    • Protocols & troubleshooting
    • Placing an order
    • RabMAb products
    • Biochemical product FAQs
    • Training
    • Browse by Target
    Company
    • Corporate site
    • Investor relations
    • Company news
    • Careers
    • About us
    • Blog
    Events
    • Tradeshows
    • Conferences
    International websites
    • abcam.cn
    • abcam.co.jp

    Join with us

    • LinkedIn
    • facebook
    • Twitter
    • YouTube
    • Terms of sale
    • Website terms of use
    • Cookie policy
    • Privacy policy
    • Legal
    • Modern slavery statement
    © 1998-2021 Abcam plc. All rights reserved.