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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?
Asked on May 02 2012
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.
Answered on May 02 2012