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Offlinejimcon202000
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Registered: 09/17/02
Posts: 177
Last seen: 14 years, 15 days
question about cloneing
    #1743088 - 07/24/03 12:23 AM (14 years, 30 days ago)

is it possible to do a clone by putting the small clean peace of "parent" mushroom right into a small container of steril grain rather then going to agar first ??  i cant do agar ... just cant do it :frown:  \
any tips
thanks
peace 


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Invisibledeanofmean
mycophagous

Registered: 12/07/02
Posts: 2,017
Loc: PNW
Re: question about cloneing [Re: jimcon202000]
    #1743346 - 07/24/03 01:48 AM (14 years, 30 days ago)

yes that will work if you keep it sterile .
but i think its easier to grow it in a liquid culture first .
i use 1 tsp. karo, 8 oz. h20, let cool after sterilization, and inject 1 cc of 3% h2o2 . you can even dip the tissue sample in 3% h2o2 for a few sec. then drop it in .
i've done this without a glovebox with good results .


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Invisiblepsyphon
mneumatic device

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Registered: 11/27/01
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Re: question about cloneing [Re: jimcon202000]
    #1743364 - 07/24/03 01:56 AM (14 years, 30 days ago)

Yes, but it may take you a few tries.  Right before you transfer the clone tissue into the jars, squirt some hydrogen peroxide in there.  Fill some sterile syringes with peroxide and put about 5cc per pint jar.  Shake each jar very well and let them sit for a moment. 

This is a good time to pick the mushroom you are going to clone.  You might want to put it into a clean plastic bag or other container to bring it to your work area. 

Flame sterilize and/or wipe with alcohol a scalpel or sharp knife.  Shave off the outer layer of the stem near the bottom like you would cut a carrot into shavings.  Cut a few slices of the stem from this area and put the pieces into your jars.

Incubate and wait. :smile: 


--------------------
"The real voyage of discovery consists not in seeking new landscapes but in having new eyes."
- Marcel Proust

I wish you all ceaselessly flowing moments of happiness.


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Offlinelemunhed
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Registered: 05/12/03
Posts: 775
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Re: question about cloneing [Re: psyphon]
    #1743861 - 07/24/03 09:06 AM (14 years, 30 days ago)

There is an exceedingly simple method of cloning at www.fungifun.org. Although on there it shows the clone tissue being placed into a petri dish of agar, you can certainly substitute any grain jar for that.


--------------------
You're pompeius, aren't you? I'll make a pompeian of you unless you hold your toungue!

-Tiberius Claudius Nero


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Invisiblemicro
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Registered: 05/09/03
Posts: 7,532
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Re: question about cloneing [Re: lemunhed]
    #1743979 - 07/24/03 10:07 AM (14 years, 30 days ago)

The only problem would be that it would grow pretty slowly this way, unless you could somehow distribute the cells throughout the grain. Otherwise, you'll have one site from where the cells will grow out. Remember to take a sterile piece from the inside, of course. I rub the outside of the fruitbody with 70% alcohol, but that's just me.

What problems are you having with agar?

--
Micro


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Offlinejimcon202000
member

Registered: 09/17/02
Posts: 177
Last seen: 14 years, 15 days
Re: question about cloneing [Re: micro]
    #1744318 - 07/24/03 12:35 PM (14 years, 30 days ago)

i just never seem to get it right ... wont gel right...dries out... contam... or will not susport growth. i give up in agar


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OfflineSeussA
Error: divide byzero

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Re: question about cloneing [Re: jimcon202000]
    #1744376 - 07/24/03 12:55 PM (14 years, 30 days ago)

Quote:

i just never seem to get it right ... wont gel right...dries out... contam... or will not susport growth.  i give up in agar 




Won't gell correctly is usually caused by one of the following:

1) weight of agar to volume of water is incorrect
2) agar not fully disolved into water
3) not using distilled water
4) sterilization temperature not correct
5) sterilization time not correct
6) using low grade agar

Dries out:

1) pour thicker plates
2) seal petri after innoculation
3) not used soon enough
4) using low grade agar

Contam:

1) improve technique
2) use flow hood or glove box  (skip the oven tek!!)
3) add antibiotics to agar mix
4) add sterile hydrogen peroxide to agar mix (after sterilization)

Will not support growth:

1) agar has dried out :smile:
2) using low grade agar
3) not adding proper nutrients to agar
4) too high temperature during sterilization causing carmalization


Remember, agar with nothing else added has very little nutritional value for whatever you are growing to consume.  You need to add sugars and starches to get things to grow.. PDA for example is agar with dextrose and potato starch added.



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Just another spore in the wind.


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Offlinejimcon202000
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Registered: 09/17/02
Posts: 177
Last seen: 14 years, 15 days
Re: question about cloneing [Re: Seuss]
    #1744934 - 07/24/03 04:28 PM (14 years, 29 days ago)

thanks seuss i will look into it


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