Mitochondrial DNA Isolation Kit (ab65321)

How should I store this kit? Before opening should I store in refrigerator (4° C to 8°C), after opening should I reconstitute the enzyme, store all components except the enzyme in the refrigerator.For the enzyme should I made aliquots and store at -20 ºC? Is it correct? I look forward to hearing from you soom. Thanks Elizabeth

ANSWER:

Thank you for contacting us.

The enzyme will be stable at 4C for short term shipping and storage (1-2 weeks at most), but we recommend removing the enzyme and storing it at -70C as soon as it arrives, even if you have not reconstituted it yet. All other buffers in the kit will be stable at 4C. Once the enzyme mix is reconstituted, it should be refrozen and it will be stable at -70C for three months.

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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Thanks a lot for your answer.

I just want to check something with you: Should be an attached document (maybe a protocol) with your answer (see below, in red)? I'm asking it because I didn't receive any attachment. In case it should be sent together with your answer, please, could you send me it again? I'm forward to hearing from you.

ANSWER:

Thank you for your reply.


The attachment referred to the kit protocol. I have attached it here for your convenience.

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

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?

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.

Hello,
I am interested in isolation of mitochondrial genome from plant tissue. Do you have any former experience/opinion of your customers in using your Mitochondrial DNA Isolation Kit (ab65321) for plants.
Cheers,

ANSWER:

Thank you for contacting us.

I can confirm that the ab65321 Mitochondrial DNA Isolation Kit has been optimized by us for mammalian samples. I am sorry it has not been tested on plant samples, and so regrettably it is not guaranteed for them.

However theoretically it can be optimized by end users for use in plants by optimizing the tissue size, cell lysis, homogenization step etc.

We aim to provide as much information as possible to customers, and I am sorry there are no more details to provide on this occasion as it has not been tested.

If you have any further questions, please do not hesitate to contact us.

Dear Sir or Madam,
I have some questions regarding your mitochondrial DNA Isolation Kit (ab65321).
The protocol from your website is only about isolation of mtDNA from cells. But I would like to isolate from brain tissue. Is this kit also suitable for this or would I need another one? Are there already protocols for the isolation of mtDNA out of tissue?
If the kit is working fine I would use this kit more often. If this is the case, is it possible to get a lower price?
With kind regards.

ANSWER:

Thank you very much for contacting us.
I am happy to let you know that the Mitochondrial DNA Isolation Kit ab65321 can indeed be used to isolate mtDNA from tissues. In fact, we previously had customers asking this question and giving us specific feedback. On the 'Scientific Support' tab on the datasheet of ab65321 you may for example find the enquiry Q4 from 24.01.2012 from someone using mammalian brain tissue. The very same customer got back to us (01.02.2012, Q2) and provided very detailed protocol information. Additional information can also be found under questions Q5, Q8, Q21, Q22, and Q23. (I hope this link works: http://www.abcam.com/Mitochondrial-DNA-Isolation-Kit-ab65321-support.html).
We basically would suggest to start with a defined amount of tissue (10-100mg) 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.
If this kit works well for you and you would like to order more of them, you could get a discount: For specific seasonal promotions (for example our 3-for-2 offers) please check on the datasheet - there should appear a banner on top if a promotion is running. Apart from this, we always offer a discount of 5% or more when you order 5 products or more. Our customer service team is happy to help you further with specific enquiries about this (030 896 779 154 orders@abcam.com).
I hope this information helps. Please do not hesitate to contact us if you need anything further.
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