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OfflineNibs
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Preferred method for genetic isolation
    #26724611 - 06/06/20 05:08 PM (3 years, 8 months ago)

Im curious to know some of the methods growers like to use to isolate genetics. The two i am aware of are:

1) Sector isolation from MS on agar, and fruiting each sector individually. I've read that you often get 10+ differnt mono cultures from spores, which is very time consuming and requires a lot more resources to isolate.

2) Germinating MS directly to grain, fruiting and then cloning the ones with the best traits. Assuming you know you have clean spores to inoculate grains  directly, this would require much less time and energy. I've read that even clones can have multiple cultures, but i would assume there would be far fewer to isolate than MS.

Now that i have a few grows under my belt, i would like to start focusing on genetics and improving my results, or even starting fresh with spores from different vendors if the overall genetics of the ones i have are weak. I have 5 strains at the moment that I would like to start doing this work on, and it would seem that cloning is the most efficient way, but i have little knowledge and experience in this area, so im interested how some of you folks go about it.


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InvisibleStrainsfordaze


Registered: 05/10/18
Posts: 669
Re: Preferred method for genetic isolation [Re: Nibs]
    #26724629 - 06/06/20 05:21 PM (3 years, 8 months ago)

Since cubes are basidomycetes they require 2 compatible monokaryons(spore germling) to fuse in order to come to recombination.
so if using spores there will be 100-1000s of strains preset.
to isolate them you would need to do successive transfer of growth until you get pure uniformity not only on agar but also with fruiting.
just because you are taking a clone doesn't mean it is a monoculture you have just reduced the number of strains.


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OfflineNibs
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Re: Preferred method for genetic isolation [Re: Strainsfordaze]
    #26724831 - 06/06/20 07:02 PM (3 years, 8 months ago)

Quote:

Strainsfordaze said:
just because you are taking a clone doesn't mean it is a monoculture you have just reduced the number of strains.




Yes, this is why I mentioned that there would be far fewer strains to isolate this way, with the added benefit of already seeing the physical traits of the strains you're attempting to isolate and avoiding a majority of the ones you dont.

Another thing I am trying to get at is that MS straight to grain without using any agar, seems like the best way to visually see all the strains the spore has to offer, and then cloning from the subsequent flushes. Correct me if I'm wrong, but it seems like a lot of potentially good genetics can be left behind when trying to clean up your cultures on agar first before spawning. Usually the quickest growing rhizomorphic mycelium is what gets chosen to make a transfer with, but what if the slower tomentose myc contains some of the strongest genes?

It seems like cloning from full spectrum MS is the most streamline way to identify and isolate the best genetics without missing the majority of them.


Edited by Nibs (06/06/20 07:04 PM)


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InvisibleStrainsfordaze


Registered: 05/10/18
Posts: 669
Re: Preferred method for genetic isolation [Re: Nibs]
    #26724871 - 06/06/20 07:23 PM (3 years, 8 months ago)

Yeah, however spores straight to grain will be a gamble if it works because spores are not sterile so incorporating them to a sterile environment is asking for contams.
Spores to agar, transfer whatever growth pattern you want til clean, it doesn’t have to be uninform just look free of contamination, then put to grain. You will still have a crazy amount of strains.


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InvisibleSmartattack
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Re: Preferred method for genetic isolation [Re: Strainsfordaze]
    #26724896 - 06/06/20 07:41 PM (3 years, 8 months ago)

You basically just need a shit load of plates, time and willingness to document your shit. Then you can pull every type of growth and fruit it all, clone everything good, repeat etc. Some people spend years on this shit. I'm 2 years into one culture hunting certain traits and am giving up to work on some others. You will end up with great stuff, but the hunt for the ultimate makes you become a fucking weirdo and in the long run just growing and taking the good with the bad is likely more rewarding.


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Invisiblemicelio
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Re: Preferred method for genetic isolation [Re: Nibs]
    #26724965 - 06/06/20 08:10 PM (3 years, 8 months ago)

My favorite way to clone:

I pull a pin that's a few hours old with tweezers and slap it on my agar dish. The growth is so fast when they're small and most of the time there's no bacteria or mold when they're that small.

I look for the fattest and healthiest pin and one that's located on top In the center of the substrate.

I end up growing each clone In bags and try to decide what ones are the keepers.



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Offlinelateforthafuture
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Re: Preferred method for genetic isolation [Re: Smartattack]
    #26724984 - 06/06/20 08:18 PM (3 years, 8 months ago)

Skipping agar is a mistake, trust me... I made that mistake..over and over. Sure you can get some clean syringes and go straight to grain and have some success...but your luck will eventually run out and you'll get very frustrated...very fast if you continue to go straight from spores to grain.

Agar's primary function is to obtain clean and healhty cultures, cloning and isolating is secondary...IMHO.

I agree it's quicker and easier to fruit, then clone, then isolate. As oppossed to doing all the isolation work up front then fruiting out each isolate.

However, this doesn't mean you get to skip cleaning up your spores on agar first.

Spores>AGAR!!!>Grain>Bulk>Fruit>Clone = Success

Spores>Grain = Fail


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InvisibleApples in Mono
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Registered: 09/21/17
Posts: 3,240
Re: Preferred method for genetic isolation [Re: lateforthafuture]
    #26725276 - 06/07/20 12:10 AM (3 years, 8 months ago)

Of course it means you get to. It's your choice. And there are plenty of people who choose to begin their isolation just as he's considering. His goal in this situation wouldn't be to minimize odds of contamination and maximize yield. He has a completely different motivation. For his purposes, there's nothing wrong with doing what he's talking about


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Offlinemushpunx
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Re: Preferred method for genetic isolation [Re: Apples in Mono]
    #26725355 - 06/07/20 12:56 AM (3 years, 8 months ago)

For what we do, honestly cloning is the better way to go IMO.


How I do strain isolation starting with MS:

I germinate my spores and start making transfers. Typically I'll need to transfer out to T8-T10 at least before I will start to see real obvious sectoring.

Next I will take a transfer from each sector on the plate, I usually pick between 10-15.

Some of those cultures will be isolates already, but often times I will have to make a couple more transfers because they are still showing sectoring.

This leaves me with 10-15 isolated strains which each need to be labeled and fruited out.
I then compare them and choose the very best fruiter.
With what we do, in addition to things like yield and growing speed, there is the added dimension of potency - which means you have to eat each of them to compare that as well.
It's a lot of work.

Out of those 10-15 isolates there might only be one or two worth keeping.


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OfflineA.k.aM
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Re: Preferred method for genetic isolation [Re: mushpunx]
    #26725811 - 06/07/20 07:28 AM (3 years, 8 months ago)

You could also dilute the spore solution and use agar.

Put a drop of a spore syringe into a shot glass with some sterile water and then stir it up and sip a swab in.

Then use the streaking technique to further dilute each section. Idk the name but it’s in c10s signature.

Taking needle biopsies instead of normal wedges has got to speed it up too.


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OfflineNibs
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Re: Preferred method for genetic isolation [Re: A.k.a]
    #26725891 - 06/07/20 08:24 AM (3 years, 8 months ago)

I completely agree that spores to agar is the safest way to go about it if going that route, and i already have 2 MS tubs on the second flush using this method. Both have very different traits and it makes me wonder how many cultures were left behind while cleaning on agar. You folks make a good point about the frustration that would result from multiple tubs over time contaminating though, which in the end makes the process that much longer.


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