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OfflinePTreeDish


Registered: 04/23/18
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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27182987 - 02/02/21 04:07 PM (2 months, 30 days ago)

Thank you sir! A few follow-up questions if you don't mind.

What does "from TYPE material" indicate?
How can you tell some of the P. rubidurum and P. parvum aren't accurately named?
You said I could just call it "Penicillium parvum group" but isn't Penicillium parvum it's own species? Why would it be denoted as a group and why wouldn't it be "P. rubidurum group" or just "Penicillium sp."?

Much of these questions come from my desire to publish accurate data myself, so it is helpful.

Finally, under what circumstances is it worthwhile to go the extra distance and use other regions to get a more clear ID? For example, I have several that don't match 100%. Isn't there some possibility that it's a new species or new strain that others might want to know about?

I do wonder how useful my work and data are to the community. Is publishing my data all that useful?


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


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27183958 - 02/03/21 06:51 AM (2 months, 30 days ago)

"from TYPE material" means it's the holotype collection, so those sequences are guaranteed to be identified correctly.  There were other sequences with the same names as the type sequences, but different sequences, so those are probably misnamed.  Or they may not be misnamed but the sequence has errors, or it's a species with a variable sequence.

See https://en.wikipedia.org/wiki/Type_(biology)

Penicillium parvum is closely related to Penicillium rubidurum, so it's equally correct to call your mold Penicillium parvum group, P. rubidurum group or just Penicillium sp.

Whether it's worthwhile to sequence additional genes depends on which other genes are available in Genbank and how badly you want to know its identity.  If the type collection has other genes available it's more important to go for the other genes than if the data from the other genes is from other collections, since those might not be named correctly. 

It's very valuable to add your data to Genbank so other people can see what parts of the world that fungus exists in.    Even if you put a name on that is wrong, others might know the right name and still be able to use the data.  It also allows other people to contact you, perhaps you have something someone wants to study.

It happened twice today that someone else found the same thing I sequenced and put in Genbank and commented letting me know that an additional collection of my find turned up.

https://mushroomobserver.org/observer/show_observation/330293
https://mushroomobserver.org/observer/show_observation/116307


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Offlinerud_dudl
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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27184285 - 02/03/21 12:43 PM (2 months, 29 days ago)

Hi PTD and AR,
I hope it's ok to jump in on this conversation. I did some DNA work on P. cyanescens and P. azurescens many years ago (like over 20) hoping to find that they were identical (but only looking at LSU & SSU rDNA). They were not identical (but there were some unusual aberrations in the P. azurescens sequences), and although I submitted the sequences to NCBI's Genbank I never published the work. In fact, I was almost kicked out of my program for doing it and took a very long break from professional mycology due to the backlash. Anyway, at the time I had a number of chats about the molecular phylogenetics trend in mycology with a good friend who is an "amateur" mycologist (who incidentally specializes in our favourite genus). He is a more accomplished taxonomist than many trained mycologists that I know. I'll never forget what he told me: there are so many people submitting incorrectly labelled sequences to Genbank because they do not have the taxonomic skills. Focusing on type specimens is a good way around this problem (if the type has been sequenced) for sequence-based IDs and phylogenetic work (but the problem remains for population-level work)...anyway, I just wish that more people took taxonomy more seriously. R_D


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: rud_dudl]
    #27185493 - 02/04/21 02:48 AM (2 months, 29 days ago)

Quote:

rud_dudl said:
I hope it's ok to jump in on this conversation.





Welcome!

Quote:

I did some DNA work on P. cyanescens and P. azurescens many years ago (like over 20) hoping to find that they were identical (but only looking at LSU & SSU rDNA). They were not identical (but there were some unusual aberrations in the P. azurescens sequences), and although I submitted the sequences to NCBI's Genbank I never published the work.




I can't find your sequences in Genbank - are they still there?

Fortunately type ITS sequences of both Psilocybe cyanescens (an epitype collection designated in http://czechmycology.org/_cmo/CM64207.pdf) and Psilocybe azurescens (holotype) are in Genbank.  I agree with your finding that they are different.


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Offlinerud_dudl
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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27186459 - 02/04/21 04:48 PM (2 months, 28 days ago)

Hi Alan,
Quote:

Welcome!




Thanks, and great to be on this site (the shroomery)...there are lots of knowledgeable people here and the reach is amazing with people from all over the world harvesting Psilocybes (and allies)...incredible opportunities for furthering our understanding of psychoactive fungi...

Quote:

I can't find your sequences in Genbank - are they still there?




They are still there, I looked on Genbank for the first time in about 20 years (!). Only, I misrepresented my work (not intentionally), I only worked with 28S rDNA with those samples. I will pm you the link. In a nutshell we found two paralogous 28S sequences in the P. azurescens sample (only one of which was posted on Genbank) which suggests a possible recent speciation event.

Quote:

Fortunately type ITS sequences of both Psilocybe cyanescens (an epitype collection designated in http://czechmycology.org/_cmo/CM64207.pdf) and Psilocybe azurescens (holotype) are in Genbank.




Excellent to have solid ID's for reference standards...

Quote:

I agree with your finding that they are different.




Most definitely different, at least from my limited work. Myself and colleagues originally thought that Paul Stamets (in naming P. azurescens) was unnecessarily "splitting" what are more-or-less identical fungi, at least microscopically.


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: rud_dudl]
    #27189981 - 02/06/21 06:01 PM (2 months, 26 days ago)

I think P. azurescens is a good species because it's quite a bit larger than P. cyanescens/allenii, stronger, and has a very limited range while these other species spread all over the west coast.

It's common for closely related species to be the same microscopically.

It's also common for different copies of the nuclear ribosomal DNA repeat regions to not be synced up.  I haven't heard that this could be due to recent speciation, but you might be on to something.


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27193469 - 02/08/21 12:50 PM (2 months, 24 days ago)

Hi Alan, I agree with you that P. azurescens is a solid species. At the time (20 years ago), myself and colleagues weren't sure.

Quote:

It's common for closely related species to be the same microscopically.




Totally, and on a related note, I am always amazed by the vast lexicon of descriptors for minute (if at all) differences in cystidia shape. Superficially what reads as totally different shapes (e.g. lageniform or sub-lageniform vs fusoid ampullaceous), may not be that different at all. 

Quote:

It's also common for different copies of the nuclear ribosomal DNA repeat regions to not be synced up.




I wish I could track down the alternate P. azurescens sequence because it was quite different from the 28S we published on Genbank (and the sample was clean, so no contaminating fungal DNA present, as far as I can remember). Anyway, if I can find the draft paper or that other sequence, I'll let you know.


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: rud_dudl]
    #27197009 - 02/10/21 11:08 AM (2 months, 22 days ago)

Here is a good reference for cystidia shape:  https://www.shroomery.org/forums/showflat.php/Cat/0/Number/9654208/an/0/page/0

Given the low variation in SSU sequences for Psilocybe, I am thinking that the highly divergent sequence was probably from a different organism.


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27198726 - 02/11/21 09:03 AM (2 months, 21 days ago)

Hi Allan, thank you for the link to the Flora Agaricina Neerlandica excerpt. I really admire Else Vellinga's work. I have used those volumes to better understand some of the NA species that conform to more European expectations (e.g. Hygrophoraceae in Newfoundland and other parts of the North Atlantic). But never for the macro- and micro-morphology drawings. Her work seems more comprehensive than the Mushrooms to Genus series which is my "go to".

I haven't had any luck locating my work (yet) on P. cyanescens and P. azurescens but when I do, I'm happy to share that info!
R_D


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: rud_dudl]
    #27213666 - 02/18/21 09:14 PM (2 months, 14 days ago)

I wanted to quickly share some advice for folks like myself who had been seeing around a 50% success rate in sequencing...until now.

Having just completed my roughly 100th sequence attempt, I recently learned that my gels weren't working right and I was misreading them. Gel not working or not being interpreted correctly == lost time+money.

For a long time, I kept seeing fluorescent bands and interpreting them as the ITS band. Unfortunately, after quite a bit of troubleshooting (and some help from Damon Tighe), I discovered that most of the items I had purchased from Odin were defective.

I had been using their anhydrous TAE buffer mix. Turns out the dry stuff, especially the EDTA, doesn't dissolve in water all that well. This was causing large pH differentials in the buffer at each end of my gel box which resulted in the gels heating up to the point of melting. The fix was to buy pre-mixed 10x TAE concentrate. No more melting.

The other big issue was that I discovered in my testing that my loading dye was throwing off fluorescent bands without any PCR product. Once I replaced the loading dye from a new vendor, my untrained eye could immediately notice how much clearer and more differentiated the bands were. Also when I realized every gel I read up to that point was suspect.

I also bought two of the same DNA ladders from Odin and neither of them worked. They would either only form a single band, smear or just not show up at all.

I'm thankful for Josiah -  without his store, the higher price-of-entry to some diybio stuff might have scared me off. But I've spoken to a handful of folks now who've also had hit-or-miss quality issues and ended up recommending buying from a different vendor. Speaking of which, I would like to highly recommend: http://bioland-sci.com/

Finally, I've been sequencing a lot of molds lately and there have been a couple that I've had repeated PCR failures. I was advised that the pigmentation in the sample can impede the polymerase reaction. To combat this, I've tried diluting the sample in water some and that helps a little. It's something to keep in mind when working with cultured fungi.


Edited by PTreeDish (02/18/21 10:49 PM)


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27214555 - 02/19/21 11:52 AM (2 months, 13 days ago)

Quote:

Speaking of which, I would like to highly recommend: http://bioland-sci.com




Have you tried contacting Josiah?

The Odin gets their products from other companies and they use it themselves often, so it's unlikely their stuff is the issue.  But if so they'll replace it.



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Offlinejzayner
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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27214673 - 02/19/21 12:51 PM (2 months, 13 days ago)

Hey, this is Josiah Zayner. CEO of The ODIN.

If anyone ever had issues with our products please contact us and we will gladly help you troubleshoot or replace your products or give a complete refund if you believe they are completely defective.

A few comments that might help with your issues

Generally, when a gel heats up it is because of resistance. A current is trying to flow between the electrodes and when there is a lack of electrolytes to carry the current or something creating a large resistance you get heat. Usually, if you are running your gel at too high a voltage(should be 150V or less generally) or current or don't have enough buffer in your gel or electrolytes in your buffer it will heat up.

We add 1mM EDTA to the buffer which is equal to about about 0.3g / liter. The Tris should make the pH above 8 and the EDTA should relatively dissolve easily. We have been using the same recipe for our buffer for years and it has run thousands of gels with no issue. I personally use the same materials we ship when running experiments and have never had issues with it. This does not to mean it couldn't have been a bad batch. If so please contact us and we will gladly replace it for free or give a refund.


The gel loading dye we use contains glycerol and bromophenol blue. Pretty much every dye on the market contains bromophenol blue(including bioland scientific). So seeing a difference between our dye is strange but maybe it was a bad batch? and we would gladly provide a refund or send you more.

Separation of DNA ladder depends on alot including the voltage used, length of time you run the gel and concentration of agar in the gel. We buy our ladder from a manufacturer and ship it exactly as we receive it. Usually ladders run strange because one used water instead of TAE in the gel or didn't run the gel long enough at a low enough voltage. Not adding enough ladder or DNA stain or method of visualization can all effect how you see it. It is possible we received a bad batch from our manufacturer and would gladly give you a refund or supply more.

As far as I can tell you never contacted us. Please do and we will gladly refund you for the products you think are defective. My team is going to perform QC again on the products you mentioned just to make sure we didn't miss something the first time.

Glad the other products you purchased from us worked great. We are doing our best as a small business to provide products at an affordable price and we appreciate your support.


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: jzayner]
    #27214827 - 02/19/21 02:49 PM (2 months, 13 days ago)

Hey Josiah, appreciate you jumping in.

Let me first say that I thank you for what you've done for the diy community. As mentioned before, I doubt I would have gotten up to speed so quickly without your store and videos. I also have a ton of respect for small businesses and I know how challenging it can be, especially in this last year. Kudos for addressing these issues head-on.

I do want to address a few quality issues that I've had. With one exception, you or someone on your team addressed it each time. Other times, I just replaced the product and that resolved my issues.

The first issue I had was with my first order. I was sent several reagents and somewhere between packaging and shipping, one or more lids came loose and the contents spilled into the zip-lock. I emailed you about it and you replaced it all, no questions asked.

On another occasion, I emailed you because one sticker said "Freeze immediately" and another instruction set said to leave at room temp. Someone on your staff clarified that for me.

I also reached out to ask if you could clarify whether the fungal ITS1F primer was ITS1 (forward) or a different primer that also happens to go by the ITS1F name but has a different sequence. A couple of times I emailed to follow up on this and no one responded. There is more color behind this confusion somewhere earlier in this thread and Alan said he would try and mention it to you directly. I never heard anything back.

I also recently ordered a refill kit and the E. coli was dead on arrival. I didn't email you because well, its possible maybe I did something wrong? But I ordered a culture of E. coli from Carolina and it worked right off the bat. So who knows what happened there.

In regards to the TAE, if it isn't fully dissolved, it will cause a large measurable pH differential at ends of the electrode and that will result in the gel melting. Is that a problem with your mix? I don't know. It could be that the TAE mix needs a bit of heat for it to fully dissolve, though I didn't see that mentioned anywhere. I do know that once I replaced it with a premixed aqueous solution from another vendor, my gels stopped melting. FYI, I use a 1% gel and I use a minipcr bluegel with fixed voltage. No issues at all with your agarose.

When I run a gel with just your loading dye (no PCR product) and the loading dye from another vendor, the Odin loading dye shows a fluorescent band, the other does not.

I've also tried two of the ladders from you and both did not perform well. Here's a video that actually demonstrates the issues with the Odin ladder (first lane) and loading dye:
https://www.youtube.com/watch?v=LIiFxp9hLEI

You can see how all of the lanes show a fluorescent band, but if you look closely (lane #4 for example), you'll see the real ITS band faintly underneath the bright band. As I mentioned before, using a different loading dye from another vendor reveals completely different results. With the Odin loading dye, I always see a fluorescent band even without any PCR product mixed in.

Quote:

Glad the other products you purchased from us worked great. We are doing our best as a small business to provide products at an affordable price and we appreciate your support.





I really appreciate you jumping in and I respect the challenges that come with what you're doing and the kinds of people (noobs like myself) you are working with.

I hope this adds some color to the conversation. And since we are both working on a similar mission to make science more accessible to all, I'm happy to help in whatever way I can to build you up - not tear you down.

Best,

Josh


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


Edited by PTreeDish (02/19/21 02:57 PM)


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Offlinejzayner
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27215109 - 02/19/21 05:42 PM (2 months, 13 days ago)

I have been this stuff for near 20 years now and have had alot of things go wrong. Some or them unexplained. I think alot of times we want to go for the simplest explaination, "It probably wasn't me so it must be the reagent." The truth is that it can be so many things that we might have missed at the time. Why I recommend reaching out to our company as we can help you with maybe something you are missing. Maybe some insight and advice from 20 years of messing up.

We quality control everything we produce in house. Extensively. Obviously, we aren't perfect we make mistakes but your supplies are one of hundreds we send out each week and the majority of those don't have issues. Again, we make mistakes. It's impossible to do anything at scale and not make mistakes.

Honestly, I have never seen anything like that gel in that video. It looks like DNA stain is moving through the gel as you can see the differential in fluorescence between the rest of the gel and the loaded lanes. Are you sure it is the loading dye and not something in your PCR reaction? Have you run the dye by itself? It could also have something to do with your excitation/emission wavelengths for your gel stain visualization. Which could also be the reason you aren't seeing the ladder? Maybe?

What excitation wavelength/ emission filter wavelength are you using? Have you been able to visualize anything yet on your gels well? Have you tried loading extra ladder? I use the same ladder we sell all the time. I have left the ladder out on the bench for months at a time with no ill effect. It's a pretty damn robust ladder. We buy it bulk from a major manufacturer so you can imagine the last thing I personally would blame would be the ladder. Though it is a possibility.

We ship freeze dried E. coli that is extensively quality controlled, like extensively because it is such a unique procedure. We remake it every few weeks even though it lasts months. We have tested how well it survives in different temperatures with the lid on and off and many other variables. So it is strange that it was dead but it is possible. Carolina ships stabs I think, so the methods for growing are completely different. If it is your first time working with freeze dried bacteria I can understand but if you contacted us we could help or ship more!

The sequence for the ITS primers are on our website page you ordered from and so can be compared to whatever paper or document you want. That was probably not responded to because of that.

I wouldn't recommend using TAE or any buffer that's not fully dissolved. Shake it and heat it until it is dissolved. It doesn't make any sense why that alone would cause a pH differential. Agarose gel electrophoresis is a continuous buffer system meaning that diffusion alone would move molecules across the gel box and so a pH differential from small amounts of undissolved buffer being located at just one side of the box doesn't seem likely. Are you sure you're not just measuring the oxidation of the electrodes or miss-measured the pH? Again, myself and lots of others use this buffer all the time and have never had issues. Same recipe for years. Not saying it's not possible that the issue is on our end.

As you become more battle worn you start to realize that it's usually not the reagents that are the problem but something else that is maybe not your fault but maybe you missed. This is why I ask to email me/us. We can help make sure you didn't miss anything even if you are an expert.

I'm not trying to rip you off or sell you defective product. We don't sell faulty products but our products might have a learning curve because we optimize for things like price, shelf and shipping stability. And if by chance something bad made it past our quality control we will definitely make it right by you. Honestly, our documentation and protocols could be better but we don't have unlimited money and humans to get that done. We are doing our best.

I just swung by this forum because someone mentioned this post and won't be back to read a response so please email me at case@the-odin.com so I can make things right. I will personally help you troubleshoot so you can have functional working products. Same goes for anyone who reads this who wants help, email me. We make mistakes but even if we don't we will try and help you.


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: jzayner]
    #27215254 - 02/19/21 07:00 PM (2 months, 13 days ago)

I answered many of your questions in my aforementioned post and some of your other responses, well, I won't get into anymore. Based on how you responded to some of what I said, you either didn't read what I wrote or we're just not understanding one another. It's cool. It's clear where you stand and let's just leave it at that.


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


Edited by PTreeDish (02/19/21 07:05 PM)


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27215988 - 02/20/21 05:28 AM (2 months, 13 days ago)

The Odin's ITS1F primer is ITS1, the F in this case means forward.  There is also an ITS1-F forward primer that is different and selects for just fungi.  ITS1 can be used to sequence plants as well, and ITS1-F can sequence the fungi in a plant/fungi mixture by ignoring the plant DNA.  Most people use ITS1-F to sequence fungi since it's more specific, but ITS1 works just as well 99% of the time.

Regarding the gel, it will melt if the resistance of the buffer is too low, causing excess current to flow.  Measuring it with an ohm meter can tell you how much current will flow at the voltage you are using.    You could also put an ammeter in series with your gelbox to see how much current really is flowing. 

The DNA migrates through the gel based on voltage rather than current, so using higher resistance buffers allows you to turn up the voltage without melting the gel.  Lower voltages give nicer gels, but when testing PCR products you are only looking for a yes/no answer, so turning up the voltage is fine, as long as the gel doesn't melt.


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: Alan Rockefeller]
    #27217148 - 02/20/21 07:26 PM (2 months, 12 days ago)

We hashed out the ambiguity about the primers earlier in the thread. For newcomers, it can be confusing to disambiguate and I just thought some sort of clarification on the page might of been helpful.

As I mentioned before, I use the MiniPCR Bluegel box which has an unmodifiable fixed voltage. After much troubleshooting, I realized that my gel was melting because my TAE mix wasn't fully dissolving. No matter how hard or how long I shook it, I could still see tiny particles suspended in solution. This was confirmed by placing a pH probe at each end of the gel box after running for a while and observing a large differential, confirming the high resistance as you mentioned.

Once I replaced the buffer with an aqueous premix, the gel stopped melting and there was no longer a pH differential. I was told the EDTA in the dry mix is known to require a higher temp for solubility. I'm not blaming the vendor or saying the only solution is to replace your buffer. I just wanted to share my learning that one should double check their buffer is fully dissolved. That was a costly lesson for me.


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OfflinePTreeDish


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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27285781 - 04/29/21 12:02 AM (7 days, 8 hours ago)

Does anyone have an issue with the borate in their TBE running buffer routinely dropping out of solution? Borate has such a high melting point, I find it annoying to have to reheat the solution to such a high temp to get it re-dissolve every time I want to barcode something.

Also, this might be too complex of a question here, but I want to learn more about generating phylogenetic trees. Is there a beginner-friendly tutorial? I don't wish to just generate a pretty graph - I'd like to understand it.

One form of question I'd like to be able to answer is: which species is a closer genetic relative to P. cubensis: A. bisporus or L. edodes? There seem to be many ways to go about solving this that are over my head. Even a "go read up on blah" would be helpful. Thanks!


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OfflineAlan RockefellerM
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Re: DNA barcoding class / interview with Adam Haritan [Re: PTreeDish]
    #27292847 - 05/03/21 09:55 PM (2 days, 10 hours ago)

I haven't had that problem with TBE, but you could also just use borax as the buffer and it actually works better.  I wonder if your concentration is too high, perhaps you are using a stock solution meant to be diluted?

See http://bitesizebio.com/25078/faster-even-cooler-dna-gels/

and https://bitesizebio.com/507/faster-gels/


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Mushrooms, Mycology and Psychedelics >> Advanced Mycology

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