[DIYbio] Endophyte isolation and first successful sequencing

[Dakota Hamill]

Hi all, just wanted to share some encouraging results from a few recent experiments we've undertaken, specifically, the isolation of symbiotic endophytic fungus without a laminar flow hood and expensive equipment, followed by sequencing data from a PCR of a common button mushroom purchased for 25 cents from the grocery store. The PCR and sequencing was basically a "validation" of our DNA isolation protocol, PCR program, cleanup kit, and sample submission, with the goal that it will be used to ID novel fungal strains isolated from plant tissue in the future.  

http://basementbiotech.org/basic-endophyte-isolation-testing-the-lower-limits-of-aseptic-technique/

http://basementbiotech.org/down-n-dirty-sequencing/

It's pretty cool to get your first chromatogram and have them all be tall skinny peaks!

FASTA sequences are in the post for BLASTing if you so desire.

-Dakota

[jarlemag]

Cool!

What concentration of chloramphenicol did you use?

-JP

[Cathal Garvey (Phone)]

Really cool, thanks Dakota!

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Sent from my Android device with K-9 Mail. Please excuse my brevity.

[Ben Gadoua]

Why wouldn't you use flame sterilization? It doesn't require any special equipment, just clean burning flammable liquid and a lighter. Just seems stupid to do this sort of thing without taking the simple steps like that. If you're going to post labbook-esque blog posts, you should include a better list of materials, components of you buffers, etc. This isn't just an issue I have with you, but a lot of the posts in diybio have some results without anything that goes with it, their procedures and materials, it really doesn't help further the community to just show what cool thing you've done but give half the details.

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[SC]

Hi Dakota,

Congratulations!  Nothing like that first DNA sequence. 

FYI, I celebrated my first one with a set of colorful stir bars.

 

Will you submit your sequence to Genbank?

 

Stacy

[John Griessen]

On 08/06/2013 10:37 PM, Dakota Hamill wrote:
>
> It's pretty cool to get your first chromatogram and have them all be tall skinny peaks!

http://basementbiotech.org/wp-content/uploads/2013/08/dd2f41d6a39848cb905f1b93b4a39a4d.png

Congrats!

[Dakota Hamill]

Thanks everyone for the feedback.

Jarl:  My lab notebook isn't at hand at the moment as I'm not home, and the plates were poured a while ago, but I think 50ug/mL.  I'l get back to you on that when I get home.

SC: Yes it was quite exciting!  Strange what things bring you joy as a scientist, as a kid it used to be candy or a new bike, now it's shipments of reagents or colorful chromatograms.  After two previous failed attempts at sequencing, it was nice to get our own data back to play around with.  I don't know about GenBank submission, at least for the short read.  We'll have to read more about the requirements for submission.  This was honestly just a quick test to try to get our own FASTA files to mess around with.  

http://dnasubway.iplantcollaborative.org/

I've been giving that software a try, though havn't had time to use all the features.

Ben: I understand what you're saying, there are times I read amazing articles or posts by people on how to build this or that and when I get to the end, I say to myself, where is all the information and materials list!  I've been quite busy during the week and have been writing these quick posts at night.  There is a section on the website that we had the intention (and still do) of filling with more detailed protocols.  Since not everyone is entirely interested (like casual interested readers perhaps) in the exact buffer recipes, perhaps what I'll do is write a detailed reference section of protocols and materials & methods in the other section of the website, and post a reference link at the bottom of the blog posts, so people that would like the nitty gritty details can get them. 

I will make a point to upload or reference the paper we got the DNA isolation buffers from, they aren't anything special, but have been working well for us and don't need silica spin catch columns.  As for flame sterilization, I don't exactly know in what capacity you are referring to.  The scalpel used to cut the tissue is dipped in rubbing alcohol and flamed before using it to excise the next piece of plant tissue.

Some people will flame the leaves after the isopropanol dip, but many others do not, and simply do a bleach wash, alcohol wash, and 2-3 water washes.  

Laminar flow hood blower and HEPA filter are on the way this week, then it's time for serious isolation!

-Dakota

[Dakota Hamill]

And also in case anyone was confused, the entire point was to grow fungus, and crowded plates with fungal colonies are what we want to see, not what most people that work with bacteria want to see!

[Dakota Hamill]

Chloramphenicol conc was 100ug/mL in plates.  Asking my friend to forward me the link to the paper he originally found the DNA isolation buffers in, I can't find it in my batch o' papers.  

[Nathan McCorkle]

On Aug 7, 2013 7:48 AM, "Dakota Hamill" <dko...@gmail.com> wrote:
>  As for flame sterilization, I don't exactly know in what capacity you are referring to.  The scalpel used to cut the tissue is dipped in rubbing alcohol and flamed before using it to excise the next piece of plant tissue.
>

That's what most people would consider flame sterilization... You specifically mentioned near the top of the post that you didn't use flame sterilization.

> Some people will flame the leaves after the isopropanol dip, but many others do not, and simply do a bleach wash, alcohol wash, and 2-3 water washes.  
>

I've heard of bleach dip, but not flaming the leaves.

[Cathal Garvey (Phone)]

Bleach dip followed by epidermal peel is sufficient in our local plant sci lab, for callous culture etc.

Sebastian? :)

[Dakota Hamill]

For leaves the bleach dip is generally all we use, but for bark the outer tissue is usually excised just because surface sterilization isn't 100% guaranteed due to all the minor cracks n crevices (not that it ever is 100% guaranteed).  The one stem samples we plated this time did not have the outer tissue excised.  Nathan thanks I fixed that line.

Not planning on publishing this in Nature, just a very crude attempt at getting some fungal DNA to sequence and cultures to look at under the microscope and perhaps broth ferment.

http://www.jove.com/video/50360/establishing-fungal-entomopathogens-as-endophytes-towards-endophytic

Hopefully we'll have a setup like at 3:51 someday!

Sebastian, do you use a laminar flow hood, or have found one that isn't $10,000?

[Dakota Hamill]

http://basementbiotech.org/endophyte-isolation-followup/

some pictures of some of the isolates, only a few turned out "pure" and those will still be further processed to insure purity.  Will try ITS PCR on them as well as finally bust out some stains and try to get some good microscopy pics.  Some of the pigmentation is really pretty!