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Offlinemistermushly

Registered: 01/11/20
Posts: 192
Last seen: 28 days, 1 hour
questions about LC / cloning / and isolation
    #26441187 - 01/19/20 05:53 AM (4 years, 29 days ago)

First of all, I'd like to thank you all for the community that exists here. It has been such a valuable resource. I've been diligently reading through the archives and have come up with some great techniques, but thought I'd directly ask the local experts here and see how that goes :smile:

I'm preparing to take on my first LC and after a lot of combing the archives these two TEK's popped out at me ...

I really like this tek from Josex 

The simplicity of this tek by creamcorn is great as well 

... I would like to take the best features of both teks, but need a bit of understanding of how LC / cloning / strain isolation / works in general, so hoping to clear some things up if possible. 

Both approaches are similar in the way that they use the needle to poke a extremely small sample of myc/tissue. I think this intuitively sounds smart to help facilitate more of an isolated strain and also seems good practice for contams. I would prefer to use creamcorn's method of taking a sample from the fruit directly.

Do you think it would be a good idea to sample from the fruit > then grow out on agar > then cut a piece to drop directly into the LC?  This seems to me to be the best of both worlds since I can take directly from the best fruiter and then grow it in agar to ensure no contams.
Could I achieve something of a mono-culture this way? Or would I have to you multiple versions of agar plates picking out sectors etc. ?
Hope that makes sense as I'm still trying to wrap my head around how to get pure strains.

My second question involves the LC recipe itself. After reading many of the threads there seem to be a few different schools of thought. Everyone pretty much agrees that the water medium ratio should be about 4% - whether using honey, Karo, LME, grains, etc. though it seems some folks believe that using something very nutritional ( and probably would grow in the LC faster ) may slow down growth when it gets introduced to the not as nutritional substrate/(rye in my case).

Thanks + I really appreciate any input.


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OfflineA.k.aM
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Re: questions about LC / cloning / and isolation [Re: mistermushly]
    #26441331 - 01/19/20 08:47 AM (4 years, 29 days ago)

I think most people go tissue to agar to lc. Dropping tissue straight in would be super risky.

I’ve made Karo lc and it worked but not great, just started an lme one. I think a lot of people go 2% even.

And no that wouldn’t be a mono culture, it would just be clone lc.

Monos take a crazy amount of work to get, and even then it’ll be a crapshoot how it performs. Clones are definitely the best method unless you’re looking for a long term project.


--------------------
LAGM2020


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Offlinemistermushly

Registered: 01/11/20
Posts: 192
Last seen: 28 days, 1 hour
Re: questions about LC / cloning / and isolation [Re: A.k.a]
    #26441852 - 01/19/20 03:24 PM (4 years, 28 days ago)

Thanks! I appreciate the info

Getting a purely isolated strain from a sample of the best fruiter sounded to easy to be true :rolleyes: I guess that's why I see some people here with enough petris to build a small house. I assume that the cloning technique would be more isolated/have less genetic diversity than multi-spore and be more likely to continue it's traits to the next generation?


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OfflineSpikeSpiegel
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Registered: 07/30/18
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Re: questions about LC / cloning / and isolation [Re: mistermushly]
    #26442082 - 01/19/20 05:57 PM (4 years, 28 days ago)

Don't skip the agar when cloning. Clone to agar then agar to LC. And test your LC again on agar before you blast  30 quarts of grain with it.

If you want to take the risk of cloning straight from tissue then read the 9er tek and adapt from that. You need good sterile technique to succeed with 9er tek but when done correctly it is a great way to clone rapidly. Here is a good place to start and the link in that post shows an adaptation of the 9er tek by eatyualive further down the post under stem tissue liquid cloning.

You also want your clone on agar so you can reliably propagate and store it after you've tested it. If it performs well you have it backed up on an external hard drive. Read this.


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Offlinemistermushly

Registered: 01/11/20
Posts: 192
Last seen: 28 days, 1 hour
Re: questions about LC / cloning / and isolation [Re: SpikeSpiegel]
    #26442622 - 01/20/20 04:39 AM (4 years, 28 days ago)

Thanks Spike - great info!

both great resources though the
verumsubsequentis tek was exactly what I was looking for to answer my questions about cloning/isolating.

cheers!


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Offlineverum subsequentis
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Registered: 03/22/16
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Re: questions about LC / cloning / and isolation [Re: mistermushly]
    #26442746 - 01/20/20 07:47 AM (4 years, 28 days ago)

Tissue sample to agar, transfer until clean (anywhere from 0 to 10000 transfers), clean myc to sterile LC ( i use 1% malt).


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OfflineMLPismyOPSEC
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Registered: 11/13/18
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Re: questions about LC / cloning / and isolation [Re: mistermushly]
    #26442769 - 01/20/20 08:06 AM (4 years, 28 days ago)

Quote:

mistermushly said:
I assume that the cloning technique would be more isolated/have less genetic diversity than multi-spore and be more likely to continue it's traits to the next generation?




That's accurate, yes.


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InvisibleSmartattack
C'mon man
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Registered: 12/21/18
Posts: 3,776
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Re: questions about LC / cloning / and isolation [Re: verum subsequentis]
    #26442786 - 01/20/20 08:22 AM (4 years, 28 days ago)

Quote:

verum subsequentis said:
Tissue sample to agar, transfer until clean (anywhere from 0 to 10000 transfers), clean myc to sterile LC ( i use 1% malt).





:lolsy::lolsy:

To OP: don't take transfer advice from guy with "wall o' plates" pic in signature!


--------------------
* Smarts videos :teacher:
* :thumbup: Planet of the APES:thumbup:
 
I'm a fungal white supremacist.


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Offlineverum subsequentis
seeker of truth
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Registered: 03/22/16
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Re: questions about LC / cloning / and isolation [Re: Smartattack]
    #26442793 - 01/20/20 08:26 AM (4 years, 28 days ago)

Yeah. Whatever you do, DON'T LISTEN TO ME!


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InvisibleSmartattack
C'mon man
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Registered: 12/21/18
Posts: 3,776
Loc: A thought
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Re: questions about LC / cloning / and isolation [Re: verum subsequentis]
    #26442798 - 01/20/20 08:29 AM (4 years, 28 days ago)

Quote:

verum subsequentis said:
Yeah. Whatever you do, DON'T LISTEN TO ME!





I've seen your cursed posts before. And because of it I now I find myself buying yet another 500 count box of plates.:judyfacepalm:


--------------------
* Smarts videos :teacher:
* :thumbup: Planet of the APES:thumbup:
 
I'm a fungal white supremacist.


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Offlinemistermushly

Registered: 01/11/20
Posts: 192
Last seen: 28 days, 1 hour
Re: questions about LC / cloning / and isolation [Re: verum subsequentis]
    #26448671 - 01/23/20 05:36 PM (4 years, 24 days ago)

good advice to ignore all your advice  :lol:

Seriously - great of you to have shared all this meticulous research and info though. super helpful! I'm starting off small - just made a handful of these for re-usable petri's that hopefully will double as LC lids if things go well.


... but 10,000 transfers to get a mono-culture?! :rolleyes:
going to have to make a few more!

:laugh:


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Offlineverum subsequentis
seeker of truth
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Registered: 03/22/16
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Re: questions about LC / cloning / and isolation [Re: mistermushly]
    #26450834 - 01/24/20 09:20 PM (4 years, 23 days ago)

Don't lose heart, my friend. I just put that in there to stress that it's an unknown amount of needed transfers. Sometimes they're clean on the first try and sometimes it seems they'll never get clean.


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