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Invisiblecarbonhoots
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Registered: 09/11/01
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cloning...why won't it work for me?
    #548579 - 02/12/02 05:18 AM (15 years, 4 months ago)

I've tried to clone twice now...all I end up with is a little blue dead chunk of mushroom on agar.

I've been taking really small pieces or strands from inside the shroom (avoiding the outside), cap and stem. It just dies on me.

Someone be a dear and type out some tips or something for me please.


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OfflineAnnoA
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Re: cloning...why won't it work for me? [Re: carbonhoots]
    #548583 - 02/12/02 05:33 AM (15 years, 4 months ago)

Do you use peroxidated agar? Too much peroxide?


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Invisiblecarbonhoots
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Re: cloning...why won't it work for me? [Re: carbonhoots]
    #549647 - 02/13/02 04:21 AM (15 years, 4 months ago)

I didn't use peroxided agar.

People talk about it like it's no big deal. Just cut a chunk of shroom...la dee da...take some material from the inside to be safe...ho hum...

It's not like that for me. It ain't working.

The chunk 'o' shroom DIES on me. Every time. I was looking forward to cloning a nice strain for once, instead of blindly picking from multi-spored agar. Now what am I suppose to do? I ain't going back to agar, you hear, I AIN'T GOING BACK!!!

Ok, ok, I'm calm


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OfflineAnnoA
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Agar? [Re: carbonhoots]
    #549664 - 02/13/02 05:24 AM (15 years, 4 months ago)

Which agar formulation do you use? What temperatures do you keep agar at?


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Invisible40oz
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Re: cloning...why won't it work for me? [Re: carbonhoots]
    #549667 - 02/13/02 05:29 AM (15 years, 4 months ago)

>>I ain't going back to agar, you hear, I AIN'T GOING BACK!!!

why not? ...agar rocks!!

im thinking its probably a "pH" problem....what do u say anno?


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InvisibleNighted

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Re: cloning...why won't it work for me? [Re: 40oz]
    #549914 - 02/13/02 12:02 PM (15 years, 4 months ago)

I agree that it's the agar that's the problem.

Carbonhoots, are you mixing your own agar or did you buy it premixed ala Fungi Perfecti or Spore Works for example?



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Re: cloning...why won't it work for me? [Re: 40oz]
    #549923 - 02/13/02 12:13 PM (15 years, 4 months ago)

There seems to be an agar problem, yes.

Don?t know if it?s pH or anything, it would be interesting to hear which agar recipe he?s using.


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Invisiblecarbonhoots
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Re: cloning...why won't it work for me? [Re: Anno]
    #551708 - 02/14/02 08:52 PM (15 years, 4 months ago)

The agar recipe is PDA (potatoe broth with 2g dextrose, 1.9g agar per100ml, plus a pinch of yeast nutrient for ph). This mixture has always done well for me with spores.

Incubated at 80-84

I checked my 6 latest tries today, and it seems 1 is growing. HOORAY! But I still don't have it down. Very hit and miss(mostly miss)

I value all your opinions, it seems to me that my failure has to do with the flesh I take from the mushroom. I need bigger chunks, or something. The cap seems to have the best flesh, but it's very thin between the top and the gills, hard to extract a nice piece. The inner stem just dosn't SEEM like nice mycelium. Quite likely I'm out to lunch on this, I've got to figure this out...

I guess I should have written I'm not going back to spores on agar if I can help it.


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Anonymous

Re: cloning...why won't it work for me? [Re: carbonhoots]
    #551889 - 02/14/02 11:39 PM (15 years, 4 months ago)

If it were contamination, you would probably see it growing at the point of innoculation. Bacteria grow Fast.

If your media worked for spores, it will work for cloning.

How big of a piece of tissue are you transfering. I get succes with very small pieces, and large pieces. Your tissue is just drying up to fast. Take a larger piece. Make sure your scalpel has cooled, cut out a piece 1mm+ big and transfer immediately. Cover and wrap your petri. As long as it doesn't dry out, it will get fuzzy white and start to grow out.

Use the mushroom immediately after harvest, don't let it dry up much before cloning.

Stick with it.
Just transfer larger pieces and make sure they are in good contact with the media.


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OfflineAnnoA
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Re: cloning...why won't it work for me? [Re: ]
    #552025 - 02/15/02 01:47 AM (15 years, 4 months ago)

Perhaps it dries up fast due to the high temperature? I keep my petris at room temperature, 71?F.


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