Re: [DIYbio] General mtDNA extraction difficulty

Dakota is absolutely correct. The major failure mode is using too much of the colony. I usually take a small tip, pick a small part of the colony into 100 ul of water, then vortex it. Use 1 ul as a template for the PCR.

On May 4, 2018, at 9:02 PM, Dakota Hamill <dko...@gmail.com> wrote:

You don't even need a kit for extraction you just boil some cells or tissue in 200uL of water for 3 minutes at 98C in a thermal cycler and use that directly in your PCR.  We've only ever done a crude boil lyse on a toothpick-head sized sample of material and have basically never had a sample fail 16S, ITS, or COX1 amplification.  That's for bacteria/fungi/fish.

For more important sample yes there are spin kits out there which are good and pretty cheap (epochbio.com I'm still using ones from 4 years ago) many people have their own personal extraction methods whether its chloroform or isopropanol etc.

Species identification via DNA Barcoding is pretty well hashed out and it's a common intro to PCR and running a gel and prepping samples for sequencing for students.

https://www.dnabarcoding101.org/resources/

https://www.dnabarcoding101.org/lab/index.html

There isn't limited availability of reagents and you don't need a lot of equipment, it's very routine and relatively cheap.  

Alternatively companies like genewiz will do all the amplification/extraction/sequencing and send you back your sequence data for $15-$18 if you just send them a colony of your bacteria.  

Would still be interested to see what species you can culture from your vinegar, or also how you make it (minus trade secrets!)

On Fri, May 4, 2018 at 11:43 AM, Reginald Smith <rsm...@supremevinegar.com> wrote:

My main experience so far has been with bacterial genetics. Given bacteria are the workhorses of vinegar fermentation the intimate details of this and related analysis and sequencing have been focused around these. I am working on a DIY Bio guide and maybe even physical kit to let people use raw vinegar from the grocery store to culture acetic acid bacteria (completely non-pathogenic), extract chromosomal DNA, PCR 16S rRNA (or several DNA housekeeping genes for a tighter match) and BLAST the results.

I got thinking about another, probably more widely interesting, kit that I am not sure is feasible. In short, it would be a kit to extract mtDNA from animal or fungal cells (not sure if you need chitinase to deal with the latter) and sequence conserved regions of cytochrome c to identify what exactly you have sampled. I know this will be a lot harder, noticeably extracting the mtDNA not just the nuclear DNA, and probably all kinds of limited availability reagents and even equipment that will add bumps on the road but is this even something feasible outside of someone like the few of us that have lab spaces and equipment? Also I am unsure if the nuclear DNA and mtDNA needs to be separated by centrifuging or chemical means so as not to screw up the PCR. Cytochrome c seems to be pretty reliable as an identifier at least at the genus level, correct? Thoughts?

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