extracting DNA from bacteria

[Towa]

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!

[cathal...@cathalgarvey.me]

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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[Koeng]

I'd recommend to just avoid gels altogether. Restriction enzymes are great, but gel purification just kills that method for me. I always gibson/goldengate, with one of the main reasons I hate cutting gels. i don't know if it is just the kit I use, but I always end up with highly salted un-pure DNA. If anyone knows of a good kit to purify DNA from gels, I'd love to know what it is. 

@Cathal I am actually going to try the method with filter paper. I will report back in a while if it works or not.

-Koeng

[Patrik D'haeseleer]

The Odin actually links to a very simple protocol for 16s rDNA PCR, from the page that you referenced:

16s rDNA Amplfiication and Sequencing

They're actually recommending to scrape a single colony straight into the PCR reaction. No separate DNA extraction step whatsoever - how simple can you get?

If you're more interested in getting the answer asap than in teaching yourself how to do 16S PCR, many sequencing services will also do 16S PCR and sequencing straight from a colony or liquid culture.

[Towa]

thanks guys! A few people also sent me private messages recommending the colony PCR route as well, so colony PCR I shall try. 

@Cathal

Thanks! I have developed a odd relationship with these bacteria. They are just so cool. The Odin tutorial on e. coli transformed with pVIB said that they like cool and dark, so I left one of my cultures at room temp at my lab desk and another in the fridge wrapped in aluminum foil, and the later was brighter. I didn't know that they "officially" don't like light and it's definitely something interesting to experiment with. In terms of the squishy gel extraction method, where does the dna end up? The Odin kit came with the green dye 

@koeng

avoiding the use of gels would help me keep the cost down haha but how do I go around confirming and cleaning out the DNA? Do the PCR clean up kit and hope there's something in there? It would be a bummer to send the result of a failed PCR reaction in for sequencing :(

@Patrick

Doh! a direct link from the page I was at... thanks for pointing it out. I think I'll take the opportunity to learn and practice lab skillz.

[Mega [Andreas Stuermer]]

http://openwetware.org/wiki/User:Andrew_Perry/Protocols/DNA_gel_extraction

I once gave a protocol like this a shot. It seemed to have worked. The DNA gets diluted but it should be possible to work with.

Attached are photographs, from when I tried it many many years ago.

[Towa]

huh, so it actually works. That's definitely cheaper than a kit. Thanks for sharing!