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OfflineBeamer710
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Cloning question
    #25964275 - 04/30/19 11:48 AM (5 years, 8 months ago)

So ive pretty much got the concept of agar down. And isolating. But one thing thats baffling me is this: how do u keep a clone that is multiple strains intact? How would one transfer without excluding any strains? Should I just cut out a donut that includes cross sections of every substrain 360 degrees around the transferred tissue? Im asking because I got a mutant jedi mind fuck in my first run of it that resembles a PE blob. I wanted to clone it and retain the mutation for novelty reasons as well as clone and isolate a well yielding culture. Thanks in advance for any input, and heres a picture of the mutant cock blob for your viewing pleasure

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OfflineShaperDreaming
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Re: Cloning question [Re: Beamer710]
    #25964301 - 04/30/19 12:09 PM (5 years, 8 months ago)

Your language is confusing. What do you mean by strain?

Take a cross section scrape near where the stipe hits the cap. If you avoid spores, everything that breaks back down into mycelium is a "clone" (I'm not sure if it's a true isolate or not). Check this out for the rundown:


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OfflineBeamer710
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Re: Cloning question [Re: ShaperDreaming]
    #25964380 - 04/30/19 01:07 PM (5 years, 8 months ago)

So what i mean is substrains. When youre looking at your agar dish and you can see all the different substrains around the circumference of growth, basically if I transfer from any specific side im going to be excluding a lot of the substrains that make up that specific mushroom i cloned. So if i want to preserve the synergy of the different substrains how can i do so while transfering from the agar to make say an LC or even just dropping wedges into grain? The only way i can think of is to cut all the way around the circumference of the culture in the agar and remove a donut shaped agar sample

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OfflineBeamer710
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Re: Cloning question [Re: Beamer710]
    #25964382 - 04/30/19 01:09 PM (5 years, 8 months ago)

Basically i dont want to include the center of the agar dish where the original tissue was transfered to, because it could host bacteria, so i dont wanna just take the entire growth, id like to use all of the different substrains but only take mycelium from the very edges of the growth. Im not very good with illustrating my thoughts with words lol

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OfflineShaperDreaming
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Re: Cloning question [Re: Beamer710]
    #25964489 - 04/30/19 02:25 PM (5 years, 8 months ago)

Ok, so you're talking once you have mycelium on agar. Got it.

Here's the scoop: unless you're a professional mycologist with a lab and expensive microscopes and shit, you're never going to hit pure isolation. It doesn't matter though as you probably won't know what you've got until you grow it out since mycelium doesn't indicate any of the phenotypes you're going after.

SO! You're going to basically be doing a bunch of guess work. You'll want to take transfers from the leading edge of the mycelium (but not around the edge of the petri dish). This transfer should be grain of rice sized. You'll want to make a transfer when the specimen is approx nickle-quarter sized. You're going to want to look for sectors that look good and likely make a few transfers at the same time.

The video clears up where to pick from to transfer and how you go about making a bunch of transfers from one initial plate. Once you have the mycelium grown out on agar you'll put that to grain and grow it out. Until you do your grow from this isolation there's no way to know if you picked the right isolate, so likely if you want to get a similar result you're going to need to grow out a bunch of bins of mushrooms and look for the one with that phenotype you like, then clone again and do it all over.

Here's a video on strain isolation that should clear some of your questions up:



Honestly, this is really complicated shit and more for fun than for producing fruits. I almost never do this unless I'm working on a clone that I really liked because you're going to spend 2-3 months chasing your tail between not knowing if you're even getting the results you want, then you may have to start all over if they aren't what you're looking for :shrug: Personally, I transfer from healthy sections until I have a "clean" plate, then put it on grains and get mushrooms. It's not the most scientific, but it's better than starting from MS syringe every-time no matter what.

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OfflineBeamer710
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Re: Cloning question [Re: ShaperDreaming]
    #25964570 - 04/30/19 03:12 PM (5 years, 8 months ago)

Thanks, im definitly still wet byhind the ears when it comes to the more advanced techniques in mycology. My thoughts on the matter were if a clone can outperform an isolate because those substrains work well together, is there a way to preserve every substrain in a culture in a single transfer? Taking a piece from the leading edge of the mycelium seems exclusive to me, there would be substrains on the other side of the circumference of the growth left behind in the transfer which seems like it would throw off the synergy of the present substrains. I suppose I could just make a slurry of liquid innoculant off the whole dish if I get it clean on the first try. Im gonna play around with this idea a bit, I'll likely post what I find/learn in a few weeks

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OfflineShaperDreaming
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Re: Cloning question [Re: Beamer710]
    #25964623 - 04/30/19 03:50 PM (5 years, 8 months ago)

Meh, isolates/clones/monocultures... seriously don't worry too much about this unless you have a super high end lab.

And, if you're going to do a LC/LI (which I don't really recommend, agar is good enough for most of our hobbiest needs) I highly recommend going with the Josex Poke Method. It will allow you to test out your growth to ensure it's clean, without adding too many vectors for contamination.

With that said, again, to avoid contamination I do not recommend working with LI/LC. After a year of constant growing I'm finally fucking around with LI/LC and it's tough to get them to not go bad on you. All attempt so far have gone bacterial.

So many people end up with issues from their inoculation cultures (agar, LI, LC, or god forbid straight up MS syringe). Over 95% of contamination comes from bad inoculation, so any extra vectors will give you chances for things to go badly. When I was really working on this stuff as a noob I would take maybe 1/4" slices of agar to inoculate my grains. It was slow, but it taught me what clean jars look like (or... very quickly taught me that I had done something wrong because contam would show up elsewhere).

Good luck!

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