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OfflineAlan RockefellerM
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DNA barcoding class / interview with Adam Haritan * 7
    #26489902 - 02/17/20 02:19 PM (1 year, 2 months ago)

Adam Haritan creates excellent nature videos called Learn Your Land (https://www.facebook.com/learnyourland) and recently  attended one of my DNA barcoding classes and interviewed me afterwards, and made a really cool video about it. 

Even mentions the Shroomery in the introduction.

https://youtube.com/watch?feature=share&v=jWP0340LCP0


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InvisibleThe_Brown_Wizard
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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #26491102 - 02/18/20 05:18 AM (1 year, 2 months ago)

:popcorn:

good stuff man


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:scaryshroom: There's a weird fuck lurking in these woods :scaryshroom:


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OfflineeLeSDenesS
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Re: DNA barcoding class / interview with Adam Haritan [Re: The_Brown_Wizard]
    #26491941 - 02/18/20 05:12 PM (1 year, 2 months ago)

nice man. Any chance you could make videos of these seminars?:smile:


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Offlinefeldman114
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Re: DNA barcoding class / interview with Adam Haritan [Re: eLeSDenes]
    #26491950 - 02/18/20 05:18 PM (1 year, 2 months ago)

Thoroughly impressed here!
:thatsinteresting::tellmeastory:


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OfflineSolipsis
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Re: DNA barcoding class / interview with Adam Haritan [Re: feldman114]
    #26492866 - 02/19/20 08:03 AM (1 year, 2 months ago)

That's really great, I noticed that video and posted it on 2 Discord servers i "officiate".
(Adam and Alan put together thats what i call something of a win-win)


Edited by Solipsis (02/19/20 08:04 AM)


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InvisibleAndyHinton


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Re: DNA barcoding class / interview with Adam Haritan [Re: Solipsis]
    #26506094 - 02/27/20 10:30 AM (1 year, 2 months ago)

Nice interview, thanks for posting. Glad to see you using the blueGel electrophoresis system, it's so compact and delightful. Your video of the class at CCL got me into community biology, which also took me away from an electronics interest. :wink:

I'm about to finish up my high fidelity implementation of this and hoped you would please weigh in on something. I have two sets of primer pairs: ITS 1F/ITS4 and NS7/LR3 according to the primer map below (source).



Given the ~1 kb read limit of Sanger sequencing and the need to trim junk off the ends, how feasible do you think it would be to splice together reads of NS7/ITS4 and ITS 1F/LR3? This is Subset 1 through Subset 2, and Subset 2 through Subset 3, with a big overlap in the region of interest. Would it run too close to the Sanger limit to produce worthwhile data in the SSU/LSU regions?

Please see the draft writeup and note that the PCR program is outdated. I must recalculate the parameters once I finalize the primer selection. I can buy more primers to sequence the subsets as shown, but it means an extra 30 assays of everything downstream of the PCR and an extra $150 in sequencing costs.


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: AndyHinton]
    #26507494 - 02/28/20 02:36 AM (1 year, 2 months ago)

Not many people use ssu anymore.    Its1f and lr3 work if DNA quality is good.  Usually people do its1f / its4 and l0r / lr7 and keep them separate. That gives a more useful part of LSU.


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Offlinedark-goblin
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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #26510521 - 03/01/20 12:38 AM (1 year, 2 months ago)

Any plans to take this to Europe? :-)


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: dark-goblin]
    #26511274 - 03/01/20 01:44 PM (1 year, 2 months ago)

Quote:

dark-goblin said:
Any plans to take this to Europe? :-)




Perhaps in November.


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InvisibleAndyHinton


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #26512534 - 03/02/20 11:51 AM (1 year, 2 months ago)

Quote:

Alan Rockefeller said:
Not many people use ssu anymore.    Its1f and lr3 work if DNA quality is good.  Usually people do its1f / its4 and l0r / lr7 and keep them separate. That gives a more useful part of LSU.



Thanks. ITS 1F/LR3 it is, then. The DNA extracts are good quality, coming from pure cultures. The PCR product should also be fine, as I'm going to use Q5 high fidelity master mix.


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: AndyHinton] * 1
    #26513477 - 03/02/20 09:19 PM (1 year, 2 months ago)

Sounds good, if you don't get amplification with ITS1F/LR3 then try again with ITS1F/ITS4.  The shorter amplicon improves chances of success - works pretty well with LR3 if I use fresh mushrooms with lots of DNA.

ITS1F/ITS4 (I use the ITS1 sequencing primer) gives about 650 bases, while a forward read with ITS1 when you ran PCR with LR3 will get you around 1050 bases, including about 400 of the LSU.  If your goal is phylogeny with lots of sequences you generate, the additional LSU can improve bootstrap values.  If you are using lots of sequences from Genbank, the additional part of the LSU won't help much because most sequences in Genbank don't have the LSU tacked on to the end of the ITS.

BLASTing an ITS sequence with LSU attached is more difficult, because even 100% matches in Genbank will have a low query coverage and therefore be scored low in the BLAST results.  Often 100% matches won't make it into your top 100 BLAST matches.  There are two ways to work around this.  One is to tell BLAST to give you more like 1000 or 5000 results, and then sort by the ident column.  The other is to use a program like ITSx to parse the LSU out of your sequence and BLAST just the ITS1, 5.8S and ITS2.


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InvisibleAndyHinton


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #26521385 - 03/06/20 11:52 PM (1 year, 1 month ago)

Thanks. It helps to have real numbers to go on, especially since I assumed 550 bp for ITS alone. The material I'm working with is pure identified mycelium, weighed out to the 330 mg parameters of the bead extraction. The production run PCRs will be with Q5 hifi enzymes.

I also quantified it at every step and want to start recording the average of three Qubit reads per sample. Then I can dilute the purified product in Tris-EDTA to the amount MGH DNA Core requires. Reading quickly at the sequencing primer guidelines it looks like the ITS 1F may be fine.

Thanks also for telling me about ITSx. My goal is to essentially to make bcrypt hashes of files on disk. I'm also looking at what ratio of saline : DMSO : glycerol would vitrify at minus 20 and have the least osmotic stress, just enough to stimulate the MAPK pathways. So a way to quickly extract ITS and make sure nobody labelled a tube wrong is great.


Edited by AndyHinton (03/06/20 11:55 PM)


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #26528291 - 03/11/20 12:29 AM (1 year, 1 month ago)

Hey Alan, congrats. After seeing the vid, I really want to take your Molecular Mycology class. It's exactly the information I've been researching and trying to piece together for the last couple of weeks.

Do you have any plans to teach a course in LA or publish one online for purchase? Could you direct me to any alternative learning sources with similar info that I could explore in the meantime?

Thanks in advance! :thumbup:


--------------------
@EverymanBio | EverymanBio Podcast | YouTube


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #26531986 - 03/13/20 03:18 AM (1 year, 1 month ago)

No immediate plans to head to southern California.  There isn't a lot of alternate learning material.

Some info I wrote on DNA sequencing is here:  https://ccl.miraheze.org/wiki/DNA_sequencing


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #26532013 - 03/13/20 04:07 AM (1 year, 1 month ago)

I found your FB group and nearly drove to Oakland to come attend the sequencing class once I found out it was the next day. :laugh:

I ended up watching the YouTube stream instead since it was cancelled. One part I really liked is how you would explain the procedure, but when you'd step away to check on something, the other guy (sorry, didn't catch his name), would add a little more background explanation behind what was actually happening.

We've got to get something going down here. The "biohackers meetup" in LA seems to be the largest semi-related group but they seem only focused on optimizing the self and not so much synthetic biology or microbiology science for that matter. I think we also have one lab that allows independent researchers but the fees are over $400/month and they don't advertise any sort of public educational training or events like you folks.


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@EverymanBio | EverymanBio Podcast | YouTube


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #26532015 - 03/13/20 04:17 AM (1 year, 1 month ago)

Hmm I don't know much about the LA biohacker scene, if they exist they are pretty quiet.


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #26537496 - 03/15/20 11:02 PM (1 year, 1 month ago)

Do you know if the CC labs wiki is down? I haven't been able to access your write-up or the dyi extract-n-amp protocol shown in the stream. Sent an email to the generic info@ but no response so far - it is the weekend and beer virus is a thing.


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@EverymanBio | EverymanBio Podcast | YouTube


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish] * 1
    #26545343 - 03/19/20 09:13 PM (1 year, 1 month ago)

It's a DNS issue, will get fixed eventually but for now you can use this address:

https://ccl.miraheze.org/wiki/DNA_sequencing


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #26545510 - 03/19/20 10:50 PM (1 year, 1 month ago)

Fantastic. Thanks.


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@EverymanBio | EverymanBio Podcast | YouTube


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #26956707 - 09/26/20 11:08 PM (7 months, 4 days ago)

I just got back my first set of sequences from MCLab and 4/8 came back all NNNN and the remainder don't appear to be usable (returned 0% match in BLAST). This was the gel.

Is there any place where I could get some help troubleshooting? I'm not on Facebook unfortunately.


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@EverymanBio | EverymanBio Podcast | YouTube


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