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Offlinezzantium2999
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LC from live tissue
    #14359194 - 04/27/11 01:13 AM (12 years, 10 months ago)

Can you make a liquid culture from live tissue out of the centre of the mushroom stem? I figured that way I can be sure to get a culture that only has one strain. Am I correct in assuming this, or is there an easier way to make sure that I'm only putting one strain in the liquid?


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OfflineBargainBab
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Re: LC from live tissue [Re: zzantium2999]
    #14359212 - 04/27/11 01:20 AM (12 years, 10 months ago)

You mean cloning? :grin: Just get some agar man :smile:


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OfflineMushaboom
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Re: LC from live tissue [Re: zzantium2999]
    #14359213 - 04/27/11 01:20 AM (12 years, 10 months ago)

It works and there are teks for it.


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Invisibleawakendone
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Re: LC from live tissue [Re: zzantium2999]
    #14359221 - 04/27/11 01:24 AM (12 years, 10 months ago)

i've never tried it, but it's been done LC Cloning

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OfflineBargainBab
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Re: LC from live tissue [Re: awakendone]
    #14359237 - 04/27/11 01:28 AM (12 years, 10 months ago)

Was looking for that link  :um:


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Offlinezzantium2999
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Re: LC from live tissue [Re: BargainBab]
    #14359246 - 04/27/11 01:32 AM (12 years, 10 months ago)

did you even read the question? I don't want to save my culture on agar. I want to make liquid culture so I can inoculate with that. The question was...Will it work with live tissue. Argh sometimes it's like people are just posting, to get more posts under there belt, and don't even read the questions. Or there are those ass holes who just post "yea I agree."' Who the hell asked if you agree. they asked a question, not if you agree with the last post. Sorry for the rant, but it just pisses me off when I need something answered and I see posts that have nothing to do with the question or topic.


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Offlinear1es
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Re: LC from live tissue [Re: zzantium2999]
    #14359393 - 04/27/11 02:40 AM (12 years, 10 months ago)

use the search engine


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Offlinear1es
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Re: LC from live tissue [Re: ar1es]
    #14359396 - 04/27/11 02:42 AM (12 years, 10 months ago)

only way to isolate is with agar

a piece from the center of the mushie will make clones but it wont be an isolate it will still contain may different genetics

or maybe im wrong


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InvisibleOmnicracker
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Re: LC from live tissue [Re: ar1es]
    #14359986 - 04/27/11 08:21 AM (12 years, 10 months ago)

it will work, but cloning, even when done right can be very dirty.  agar will make sure you didnt get any contams.  if you go to LC direct, it could get contamed and you'll never know.  it will work though.  just put 2 marbles and some shards of glass in the jar to help break things up when it gets growing.


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OfflineTrippy_Smurf
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Re: LC from live tissue [Re: Omnicracker]
    #14360037 - 04/27/11 08:37 AM (12 years, 10 months ago)

You'll get attitude anytime you ask a question that has been answered 100's of times and can easily be found with the search engine.

You can clone the stem, which will only have one set of genetics, but there's a good chance the LC will be dirty/contaminated since the tissue sample is not sterile.  Agar is used to make sure you're working with a clean sample before you waste your time and resources.

If you're going to skip the agar, make sure to noc up a few pf jars to test it before you use it on lots of jars.


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InvisibleDoc_T
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Re: LC from live tissue [Re: zzantium2999] * 1
    #14360042 - 04/27/11 08:39 AM (12 years, 10 months ago)

Quote:

zzantium2999 said:
Or there are those ass holes who just post "yea I agree."' Who the hell asked if you agree. they asked a question, not if you agree with the last post.




Agreed.


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InvisibleSillicybin
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Re: LC from live tissue [Re: Doc_T]
    #14360104 - 04/27/11 08:56 AM (12 years, 10 months ago)

Quote:

Doc_T said:
Agreed.




Doc_T for president. :biggrin:

Can you go from live tissue to LC?  Yes.  Should you?  No.  You will almost certainly get an LC with bacterial contamination.  LC is a contamination magnet.  It's awesome when it's sterile, and a huge waste of time when it's not.

A better way to do this might be to toss a good biopsied fruit chunk into a grain jar using a glovebox.  With that, you will have better chance of success because even if it does have some contamination on board, the mycelium might be able to overcome it in grain where it certainly cannot in LC.  Then you can use Doc_T's method of making GLC once you've got a solid grain jar.

All of the above is the slacker way of doing things.  If you really want to clone the right way, procure and learn to use agar.

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InvisibleDoc_T
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Re: LC from live tissue [Re: Sillicybin] * 1
    #14360109 - 04/27/11 08:57 AM (12 years, 10 months ago)

Quote:

Sillicybin said:

Can you go from live tissue to LC?  Yes.  Should you?  No.  You will almost certainly get an LC with bacterial contamination.




Agreed. I never did get it to work, although lots of people do.


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InvisibleDoc_T
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Re: LC from live tissue [Re: Doc_T]
    #14360113 - 04/27/11 08:59 AM (12 years, 10 months ago)

Also, a clone is not an isolate. Generally there will be several, possibly many, substrains in a tissue sample.


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Invisibleanonjon
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Re: LC from live tissue [Re: Doc_T]
    #14360126 - 04/27/11 09:03 AM (12 years, 10 months ago)

I've dropped colonized popcorn grains, bits of colonized substrate, core tissue, pins, agar into liquid culture with success.

There's just a lot of failure that goes with it. So u make several and toss the ones that turn cloudy.  Core tissue and agar can be used without a peroxide dip, but any other kind of tissue transfer to lc is going to need to be sterilized in h202. If you go over 20 seconds tho there is a tendency to kill the tissue.

The reason people are encouraging u to go to agar as an intermediate step is to increase your chances of success. They aren't trying to be dicks.

If you do go tissue -> sugar lc , then make several at one time cuz some will probably turn on u. Remember that even the slightest cloudiness means it's toast.

GL!


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InvisibleSillicybin
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Re: LC from live tissue [Re: anonjon]
    #14360148 - 04/27/11 09:09 AM (12 years, 10 months ago)

anonjon - I'm pretty sure that post right there has to be the nicest-toned one you've ever submitted.  I'm ... shocked.... Keep this up and we're going to wonder if you got kidnapped and replaced by someone else. :tongue2:

All of the suggestions are spot-on, exactly my experience with it.  The H2O2 dip is extremely helpful in trying to clean up a tissue sample for LC - that's the only way I was ever able to get it to work, and even then I still got over 50% failure rates.

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Offlinegornyhuy
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Re: LC from live tissue [Re: Sillicybin]
    #14360174 - 04/27/11 09:18 AM (12 years, 10 months ago)

To take it one step farther, if you want to have some fun, do it right, and get great results:

1)tissue -> agar
2)clean & isolate your clone on agar
3)agar wedge to one jar of grain
4)make a grain LC "GLC" from that jar
5)inject inject inject!!!

Now you are pretty much guaranteed to be 1)cloned 2)isolated and 3)clean as a whistle.


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OfflineTrippy_Smurf
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Re: LC from live tissue [Re: Sillicybin] * 1
    #14360230 - 04/27/11 09:34 AM (12 years, 10 months ago)

Quote:

Sillicybin said:
Quote:

Doc_T said:
Agreed.




Doc_T for president. :biggrin:






I agree.


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Invisibleanonjon
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Re: LC from live tissue [Re: Sillicybin]
    #14360973 - 04/27/11 12:57 PM (12 years, 10 months ago)

Quote:

Sillicybin said:
anonjon - I'm pretty sure that post right there has to be the nicest-toned one you've ever submitted.  I'm ... shocked.... Keep this up and we're going to wonder if you got kidnapped and replaced by someone else. :tongue2:




It's all my new gf's fault. She's makin me soft!


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Offlinek00laid
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Re: LC from live tissue [Re: zzantium2999]
    #14361001 - 04/27/11 01:03 PM (12 years, 10 months ago)

agar is for more than saving your cultures, OP..


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