Insect mitochondrial isolation

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Insect mitochondrial isolation

Postby corto » Mar 15 2012 12:21 pm

Hi, just wondering if anyone has heard of a good protocol for isolating mitochondria from whole insects. I'm currently trying to sequence the mitogenome of an insect of interest and I realize there are (at least) two ways to go. Most people seem to choose extracting genomic DNA and performing mitochondria-specific long-range PCR to get sequence out. I was thinking it should be possible to isolate the mitochondria and then perform rolling circle amplification, but I haven't been able to find much on the matter. Mitochondrial isolation procedures are easy to find for rat liver's etc. so maybe I could adapt one of these for some pooled whole body insect samples?

Thanks in advance!
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Re: Insect mitochondrial isolation

Postby biochembug » Mar 15 2012 7:04 pm

What sample you are handling, Insect cell line or actual insect tissue?

You may be able to isolate mitochondrial fraction for a cell line (requires optimization), however, I won't try it for my precious samples such as actual insects. PCR is a proven boon for them. My colleagues from a previous lab used to do PCR after extracting total DNA from insect parts.

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Re: Insect mitochondrial isolation

Postby corto » Mar 28 2012 2:38 pm

Working with whole insect specimina, and I've come to the same conclusion that PCR is the way to go. Putting my effort into optimizing long PCR's. Thanks for the input!
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Re: Insect mitochondrial isolation

Postby biochembug » Mar 28 2012 5:54 pm

For long PCR, I would recommend you phusion (http://www.neb.com/nebecomm/products/productm0530.asp). Do not waste your time with Pfu. I have amplified plasmids upto 10 kb.
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