Mitochondria are semiautonomous 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. The Mitochondrial DNA Extraction Kit 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.
|#Cat + Size
|K280-50 (Size: 50 assays)
|Enzyme manipulations, Southern blotting, cloning, PCR analysis, and amplifications.
|Features & Benefits
| • Simple procedure; takes less than 2 hours
• Fast and convenient
• The kit provides unique formulations of buffers and reagents for isolating a highly enriched mitochondrial fraction. Mitochondrial DNA are then isolated using reagents included in the kit
| • 5X Cytosol Extraction Buffer
• Mitochondrial Lysis Buffer
• Enzyme B Mix (lyophilized)
• TE Buffer
|For Research Use Only! Not For Use in Humans.
What is the temperature needed for the enzyme inactivation of enzyme mix?
Enzymes in the enzyme mix can be inactivated by heating at 95 °C for 10 min.
Is there a need to degrade the contaminated RNA in mitochondrial DNA with RNase in additional step after using this kit?
The lysis buffer has RNase added to it. Therefore, you don’t need to add RNase additionally.
How do you determine the purity of mitochondrial DNA?
The circular mitochondrial DNA runs ~15-20 kDa on agarose gel, whereas genomic DNA runs much big size. The size difference can easily be used to differentiate between the two. You can try different enzymatic digestions also to see the difference. Here is a whole article showing you the differences between mt and genomic DNA: http://www.sfrbm.org/frs/Ballinger.pdf
I got a smear pattern, begin nearly 22KDa. Why?
"It might happen due to the cells containing high levels of endonuclease. You add endonuclease inhibitor into the buffer system at beginning homogenization step, where the DNA degradation may occur. In addition, it may help if you add 5 ul enzyme B Mix immediately after lysing the mitochondria without keeping on ice 10 min. Decrease the time to lyse the mitochondria may decrease mtDNA degradation, since Enzyme B contains Proteinase K which will kill all endonucleases."
Does this kit work with tissues?
Tissues are much more dirtier than cultured cells, and very sticky after homogenization. There will be genomic DNA contamination in the mDNA prep. Also mDNA may get partially degraded by DNases. You need to do some modifications to isolate mDNA from tissue: 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 keep on ice for 10 min, and directly go to step 12, add 10 or 15 ul Enzyme B Mix, then put in 50 degree C overnight. Enzyme B mix will degraded all proteins and DNases.
What is the mass at the bottom of the gel and how to get rid of it?
It is RNA. The mitochondrial lysis buffer contains Rnase. After step 15, when you collect the pellets of mitochondrial DNA, wash with the mitochondrial lysis buffer and precipitate again.
What is the mass above 16 Kb on the Gel? It is genomic DNA.
What is the Kb for Mitochondrial DNA?
The Kb of mitochondrial DNA on agarose gel runs ~16-20.
Do you have any information about the expected mtDNA yield when 10 mg of tissue are extracted?
~5 - 20 µg mtDNA can be isolated from each mg of tissue.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact your regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
I am planning to use mouse tissue for DNA extraction? Do you have a protocol for tissue samples?
Please use the same protocol that is used for cells to process tissue samples as well. Please use 50-100 mg tissue/sample. You can start from Step IV-3 under the “Mitochondrial DNA Isolation Protocol” section.
Can you describe the differences between K389-25 and K280-50?
K389 Mitochondrial DNA Isolation Kit facilitates purification of high quality mitochondrial DNA from mammalian cultured cells or tissue. A density gradient separation method is utilized to isolate mitochondria from the rest of the cellular organelles. This is followed by an enzymatic reaction to release DNA from the mitochondria. Upon release, mitochondrial DNA is further purified from nuclear DNA contamination and adsorbed onto a silica spin-column under chaotropic conditions, eliminating the use of toxic organic compounds or solvents. K280 is not based on density gradient separation of the mitochondria, but it is a reagent based methodology. For K280, ~5-20 µg of mtDNA can be isolated based on the starting material. For K389, ~2 -20 µg of DNA can be obtained based on the starting number of cells/tissues as specified in the data sheet.
1. Mo Wang, Characterization and Regulation of Carrier Proteins of Mitochondrial Glutathione Uptake in Human Retinal Pigment Epithelium Cells. Invest Ophthalmol Vis Sci, Feb 2019; 30707752.
2. Xin, G. et al., (2017) Xanthohumol isolated from Humulus lupulus prevents thrombosis without increased bleeding risk by inhibiting platelet activation and mtDNA release , Free Radical Biology and Medicine, Jul.2017, 108:247-257
3. Cividini, Federico et al. (2016) O-GlcNAcylation of 8-Oxoguanine DNA Glycosylase (Ogg1) Impairs Oxidative Mitochondrial DNA Lesion Repair in Diabetic Hearts, J Biol Chem. 2016 Dec 16;291(51):26515-26528.
4. Pierini et al., A Tumor Mitochondria Vaccine Protects against Experimental Renal Cell Carcinoma. J. Immunol., Oct 2015; 195: 4020 - 4027.
5. García-Ruiz et al., In vitro treatment of HepG2 cells with saturated fatty acids reproduces mitochondrial dysfunction found in nonalcoholic steatohepatitis. Dis. Model. Mech., Feb 2015; 8: 183 - 191.
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