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 deathblade
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 A cloning Question
#11232062 - 10/12/09 08:45 AM (2 years, 4 months ago) |
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I prepared the petri dishes and yesterday I put a few little chunks onto them. The technique I used was to pick a couple of shrooms that werent fully grown and then put them into my glove box. I ripped them in half and picked a small chunk from the inside of the stem and put it onto the agar. Will this work? Or is there a different method to be used when dealing with P.Cube?
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cloudsaregathering
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no thats how it is done just did it the other day as a natter of fact...
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deathblade
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Thats good to hear. How big of a piece did you use?
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cloudsaregathering
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i use about the size of a pencil lead, thats in a 60mm petri dish also... but nothing to large is needed...
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deathblade
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How long? As long as a pencil? Mine are only 1cm long.
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 mycoelf
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hello,
When cloning P cubensis I have developed the habit of finding a really nice young specimen with veil intact, I wash the shroom gently using a soft bristle brush to remove and casing material, and then I spray the clone with a solution of H2O2. Let fissile until the reaction stops.
Then in sterile field I split the shroom in half lengthwise looking at the ropey mycelium in the interior of the core. When I have an intuition about the best section (usually near the base) I take a cross section in front of and behind the target section and remove the strand usually no longer than 1/4 inch and place it in the petri.
Since I have done the prewash and peroxide treatment I have found it easy to pull clean clones. I usually take no more than 3-5 60mm dishes finding success in that number. If I were coloneing something precious of rare I would do no less than 10 petri dishes. If I were to be absolute I would do 20.
Nothing is more frustrating than trying to clean up a desperately needed clone, so the ounce of prevention is worth the LB of cure.
I also think it is important to cool the scalpel blade in the receiving petri as this fluidizes the agar and helps that inocculum get hold.
Many blessing of the little children upon you
MycoElf
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Mycoelf
I love to trade wedges. Currently looking for a nice brick top and Pleurotus Eunosmus the Tarragon Oyster. I am also getting interested in the agaricus, so if you have a cool agaricus in good genetic condition PM me please
Sterility is a process that can be likened unto infinity, which is a long walk, the closer to the end you start before beginning, the more achievable the goal of infinity becomes. Remember, cleanliness in next to goddessness
   
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anonjon
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Re: A cloning Question [Re: mycoelf]
#11235629 - 10/12/09 07:21 PM (2 years, 4 months ago) |
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I have a similar technique that hasn't failed yet.
I put the shroom and the agar jars and a small jar of peroxide water in the glovebox.
I have an alcohol candle in the gb as well.
I flame the scalpel and a small pair of tongs.
I cut of the cap of the mushie, then slice off a circular cross section of the stalk just under the cap and let it drop into the peroxide.
I let it fizz for a moment and then grab it with the tongs and drop it into the agar jar.
Is pretty simple and effective.
(homemade alcohol candle, 'scalpel', vegetable tongs, popcorn w/ agar wedge, 1/4 pint agar jars with polyfill breather)
Edited by anonjon (10/12/09 07:39 PM)
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RogerRabbit
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Re: A cloning Question [Re: anonjon]
#11238570 - 10/13/09 09:13 AM (2 years, 4 months ago) |
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If you're cloning indoor mushrooms, there's no need for the peroxide. As long as you're getting virgin tissue from the interior of the fruit, it will be clean. Outdoor fruits are a different story, but generally indoor mushrooms are very clean. I've even dropped whole pins on agar and had them grow without contamination.
RR
 A few days later ------> 
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deathblade
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All I did was make sure that I didnt touch it with my hands and that the samples were from the very centre of the stem. I looked at them today and can see signs of growth on all the pieces. Including the ones that oxidized quite badly.
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fastfred
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IME it's a good idea to prep samples with peroxide and bleach, even if they're indoor and you're taking clean tissue from the center.
I usually take a stem and dip it in peroxide for 5 min, then 1:10 bleach for 3-5 min. Usually I only do this for the outside then take tissue from the inside, but you can do this with inner tissue also without killing it.
Also, some of you don't seem to be getting the idea of why you're doing this in the first place. Don't use immature fruits, because then you have no idea how they would have developed. Only clone the best fruits at their peak. It's also a good idea to let them get past their peak so you know things like how long it takes for the veil to break, how large they get before and after the veil breaking, etc.. You're not always going to catch them exactly at the right point so it's a good idea to choose fruits that will perform well under varying circumstances.
-FF
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anonjon
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Re: A cloning Question [Re: fastfred]
#11240898 - 10/13/09 04:22 PM (2 years, 4 months ago) |
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The only trait I've been interested in cloning is clustering. But I'm working with pe and haven't noticed much diversity in the fruitbodies.
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RogerRabbit
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Re: A cloning Question [Re: anonjon]
#11243063 - 10/13/09 09:20 PM (2 years, 3 months ago) |
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I disagree fred. By capturing pins or young mushrooms, you capture the vigor of young, rapidly dividing cells. The growth and performance of a cloned pin are far superior to a cloned mature mushroom.
RR
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anonjon
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Quote:
RogerRabbit said: The growth and performance of a cloned pin are far superior to a cloned mature mushroom.
RR
Are you certain on this? I'm not challenging you, I'm just wondering if I need to change my method and start cloning younger fruits. I've been doing fairly mature ones.
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RogerRabbit
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Re: A cloning Question [Re: anonjon]
#11246956 - 10/14/09 02:49 PM (2 years, 3 months ago) |
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Yes. I've done it both ways numerous times. Mycelium from pins grows and fruits faster than mycelium from mature mushrooms, even when stored on master slants for years.
RR
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Morelman
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I agree with both RR and FF. Each have a valid point and cloning both stages of development have their place in culture work. There are drawbacks to both that can't be helped.
Right before the veils start to rip. You have a pretty good idea which fruits have the genetics you're after. No need to let them mature past that point.
If you want young, vigorous, dividing cells for storage. Fruit the clone first and THEN collect the pins from the cloned flush. Yes, they may be a little closer to senescence. But, at least you'll already know your sub-strain's genetics without having to wait until it reaches maturity. A workable compromise.
To get an aggressive sub-strain. Use first flush fruits whenever possible. 
Edited by Morelman (11/18/09 08:11 PM)
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fastfred
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Re: A cloning Question [Re: Morelman]
#11252045 - 10/15/09 09:20 AM (2 years, 3 months ago) |
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Quote:
I disagree fred. By capturing pins or young mushrooms, you capture the vigor of young, rapidly dividing cells. The growth and performance of a cloned pin are far superior to a cloned mature mushroom.
RR
That's a good point. But I hope people are working on a breeding program. If you're just looking for a good culture for production then you might want the youngest cells possible. But if you're breeding them it's really just an intermediate step.
It's just too easy to grow them, so I never worried about quick production. If I were a big production house I probably wouldn't be using cloned tissue except for breeding purposes.
-FF
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anonjon
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Re: A cloning Question [Re: Morelman]
#11252054 - 10/15/09 09:21 AM (2 years, 3 months ago) |
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I dunno what traits you'd really care about cloning for that you would need to see full development.
If the younger ones are that much more vigorous, it seems like the way to go.
Specially since clustering is a good trait to clone for and it's apparent very early.
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deathblade
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Re: A cloning Question [Re: fastfred]
#11252065 - 10/15/09 09:23 AM (2 years, 3 months ago) |
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Do my clone dishes need light? I have them in the same place as my jars.(warm and dark)
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cloudsaregathering
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i've never put mine in the light...
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deathblade
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My only get light when I take them out to look.
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anonjon
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Mine colonized in the light and I saw no noticeable difference.
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deathblade
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Re: A cloning Question [Re: anonjon]
#11259353 - 10/16/09 10:28 AM (2 years, 3 months ago) |
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It has been 5 days. All I can see is rhizomorhic growth. There is only a little bit of fuzz growing on the pieces. It looks like most of the growth is in the agar, not on the surface. I would put up pics but my camera doesnt work well enough. I think that I can see some sectoring. How long would you guys leave it before starting the next transfer?
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anonjon
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If one sector is big nuff you could just take out a piece and inoc a grain jar now. Then go to grain lc when that jar colonizes.
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deathblade
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Re: A cloning Question [Re: anonjon]
#11259586 - 10/16/09 11:09 AM (2 years, 3 months ago) |
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I am trying to isolate it. I have more jars started so I am not worried about losing the strain.
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anonjon
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I dunno why you'd want to wait before you transfer it again. Go for it.
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SirBeatNikCat
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Re: A cloning Question [Re: anonjon]
#11370469 - 11/02/09 03:00 PM (2 years, 3 months ago) |
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I wanted to make a comment on when they are to be cloned:
If you take them as they are smaller, when they "vigourous," it wont make the cloned shroom vigorous, just because.
if you take it when it is fully fruited, and you can see the results, then you know the way it fruits and such.
Either way you will get the same result, from the same fruit.
Im not a expert or shroomin like those here, But I do know genetics.
DNA is DNA.
If you take a clone of a rose in the spring because it grows vigorously, and then expect the clone to do the same, it wont be true, unless you give it the right enviroment to do so.
I think I would wait then take the clone when the results were known.
peace,
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Mycelio
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Yes, DNA is DNA. The difference I see, lies in epigenetics, in other words in the activation pattern of the genes. So when taking a tissue sample, which is currently in fast growing mode, mycelium will grow faster, compared to a sample, which already stopped growing or - in the worst case - entered dying mode after the fruit body reached full maturity. So it has to change gene activation, before it can start growing, while in the first case it just continues.
Carsten
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anonjon
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Re: A cloning Question [Re: Mycelio]
#11375970 - 11/03/09 09:37 AM (2 years, 3 months ago) |
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Is that why i couldn't clone myself by putting my boogers on agar?
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Mycelio
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Re: A cloning Question [Re: anonjon]
#11376329 - 11/03/09 10:39 AM (2 years, 3 months ago) |
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Yes, better use a large biopsy needle and extract a tissue sample from the inside of your balls, alright?
Carsten
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cloudsaregathering
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Re: A cloning Question [Re: Mycelio]
#11386696 - 11/04/09 06:01 PM (2 years, 3 months ago) |
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whoaaa... boys...
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SirBeatNikCat
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Mycelio,
If I am to understand If I took a clone from a little shroom that was growing quickly,( and if I could,) took another clone of it when it was mature, then I would see two different growth patterns,
One quick, from its youthful vigor, and the other slower,because of age.
But both contain the exact DNA.
I know from cannabis that from seed you get way better growth then from clones.
because the clones are of an older age, and dont have that vigorous stage they had at their beginngs from seed.
I am certainly no expert on this, as with shrooms I am somewhat guessing at best.
But it seems that when mycelium begins to produce shrooms thats its "vigorous stage."
However from a fully fruited shroom, it would be passed that stage, however when the clones where fruited they would hit the vigorous stage same as with any other all others at that point.
But I dont think you can capture that stage for good by cloning young.
cause then you would have to hope they also fruit big as well, fast.
Plants and shrroms are two different things, so I could be totally off on this.
But I would like to know.
If its possable to capture the activation pattern, to pruduce faster, Thats great to know.
I would like to know more about it.
Mr K@
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Mycelio
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Hi, I wasn't talking about age, that is a different story. What I meant is the current state, the mycelium is in. At pin stage, there is fast growth going on, while at mature stage this growth has stopped already and a few days later the mushroom tissue would start to die. I am sure there will be a big difference in growth during the first days after cloning, but I don't know for how long there will be a difference. I also don't think that you can capture that speedy or slow growth for the rest of the cultivation, but when starting with vigorous tissue, the next grow may be completed one or two weeks earlier.
If you are doing a multispore grow, you better wait for the mushrooms to reach full size, just to determine, which ones you like most. As RR, FF and Morelman discussed this earlier in this thread, each has its pros and cons.
For plants, everything seems to be different. While mushrooms and animals seem to be counting cell divisions by cutting of telomeres from the ends of the chromosomes, plants seem to be able to reset this mechanism if multiplied by cuttings or grafting.
Carsten
Edited by Mycelio (11/08/09 09:49 AM)
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