Re: [DIYbio] extracting DNA from bacteria

Congratulations on your new glowing minions! :)

Watch out for a small gotcha with V.phosphoreum; at least with the strain that I isolated years back, when grown under light they do not glow. They need to be insulated from the light to produce the luciferin. Whether this was photobleaching or transcriptional regulation, I do not know; would make an interesting (and perhaps publishable) project, though. Even finding out which frequencies it responds to would be fun.

For DNA extraction, you're dealing with a gram-negative without much peptidoglycan, so there are plenty of handy options. Classic alkaline lysis will work, as would ultra-dirty "boiling lysis" methods, and in either case you should get plenty of 16S to work with IMO. I won't comment on the procedure for 16S identification itself because I've never done it. Just make sure you clean up any detergent and salt from your prep method; dishwashing detergent for example may have tons of unidentified enzyme inhibitors, and good luck getting the company to tell you what's in it! Ethanol is apparently better than isopropanol for de-salting DNA after extraction, I'm not sure how it compares on detergents.

I'm *told* that the best gel extraction method is to slice your DNA fragment out as small as you can make it, then put it in a PCR tube with a hole cut in the end and some filter paper covering the hole..then spin it inside a larger tube in a centrifuge, so the gel fragment gets squished flat and the liquid escapes into the larger tube. I have also never done this, however. :)

The best advice I can give for gel extraction is..never, ever use Ultraviolet. Not even once. Go blue or go home. :) So, SYBR dyes or similar would be fine, but an Ultraviolet dye will lead to shredded DNA and poor PCR outcomes.

August 30, 2016 5:27 AM, "Towa" <tow...@gmail.com> wrote:

hey guys! I was able to culture some bioluminescent bacteria from squid and I want to know what it is. My guess is that its V. Phosphoreum but to confirm, I want to extract it's DNA and try the 16S rRNA identification with primers from Odin (http://www.the-odin.com/bacterial-16s-rdna-primers-for-bacterial-identification). Should I be able to get PCR-able DNA out with dish detergent and ethanol/isopropanol? Any good protocols that are DIY friendly?

on a side note, does anyone have either a) good DIY gel purification method or b) someone that will sell kits to ronin-hackers (do not belong to any institution)?

as always, thanks!

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