Re: [DIYbio] Endophyte isolation and first successful sequencing

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.  

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