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Clone Selection and Agar Propagation for Dummies


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#1 fedshtkpndrk

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Posted 08 March 2008 - 01:55 PM

Clone Selection and Propagation of Agar Media for Dummies

I started out in this hobby enamored with cloning, curious as to the process and then turned off by the level of detail and sterility that conventional techniques demand. I dislike glove boxes a lot, I cant stand wearing gloves while working either. I wanted to find a cloning tek that was simple and could be done in open air, although it seems like the Holy Grail. I was sent a cloning tek by Livewire420 and was asked if I could check it out, I immediately fell in love with the tek and tried it out. This is a syringe coring tek, just like a biopsy. Slow as we go, I’m going to stuff this thread with detail. I will do my best to document the process in real time, so bear with me :).

Days 1-21 Were spent preparing spawn and substrates Inoculating with multispore LC or Spore Solution and allowing for colonization (many examples can be seen here in the Mycotopia Web Archive: Grow Logs or Mushroom Photo Gallery & Grow Logs - Mycotopia Web Forums . Strains are PF Albino and Penis Envy both from Sporeworks.

PFA pins

http://mycotopia.net/forums/attachment.php?attachmentid=79813&stc=1&d=1204998881




(03/08/08)

Substrates were late cased with a 60/40 Verm/Coir casing layer. In the next few days we will begin to see the MS fruits developing, We will decide which fruits to clone based on several parameters, pinning speed, thickness, length, and overall aesthetics. I will place all Syringe core clones on antibiotic agar in ½ pint jars. All clones will be done open air, I will expect a couple to fail as I will be trying to work the camera at the same time. I have had an 85 percent or so success rate overall with learning curves using this tek.



I hope I can help the peeps out there who cant afford a flowhood, or are simple like me and dont have room for any more equipment :)



Texan Clone


http://mycotopia.net...=1&d=1204998881





-feds

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#2 tomstevens

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Posted 08 March 2008 - 03:10 PM

Lookin good feds...good luck thanks for the info...:headbang:

#3 the_chosen_one

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Posted 08 March 2008 - 03:36 PM

gonna be a good one!

*grabs seat

#4 eatyualive

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Posted 08 March 2008 - 09:18 PM

looking great...

also, the 9er tek cloning method is definitely up there in speed and simplicity.

to me hands down 2nd to none but i haven't read newer teks bc it worked so well, but it isn't isolation either. its more selective cloning.

can't wait to see how this turns out!

#5 fedshtkpndrk

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Posted 13 March 2008 - 10:09 AM

First off I would like to show you the 1/2 pint jars I use, there is nothing really special about it. The lids are straight from Hippie3's Easy LC tek and airport syringe :D I am using antibiotic agar, but any agar should work.

http://mycotopia.net...=1&d=1205416919

I checked on the PF Albino last evening to find the caps opening on a couple. I decided that this would be a great time to take some pics of the clone selection of the PF Albino. I started off with a light squeeze on the largest fruits. I found the the one I had been eyeing all along to be the most dense and second largest. I peeled the fruit away from the other.

http://mycotopia.net...=1&d=1205417201

***THIS IS DONE OPEN AIR OBSERVING BASIC STERILE PROCEDURE***

I Started with a simple needle coring through the stem. First by applying 70% rubbing alcohol around the area of the biopsy. I quickly found this to be ineffective with the soft tissue of the PF Albino. This only created an ugly fruit. On a side note this is my first time working with PF Albino and I'm amazed at the quickness of the bluing reaction.

http://mycotopia.net...=1&d=1205417201

Instead of totally butchering the fruit body, I decided it would be best to get the tissue from the cap on this particular strain. I swabbed the cap with 70% isopropyl alcohol and took a Needle core at a 90 degree angle to the fruit body through the peak of the cap.

http://mycotopia.net...=1&d=1205417201


As you can see the sterile clone was taken just prior to these examples. Here is the Core inside the needle.

http://mycotopia.net...=1&d=1205417201

Here is the core after is was shot out onto my finger.

http://mycotopia.net...=1&d=1205417201



And finally there is the core safely on the agar.

http://mycotopia.net...=1&d=1205417201

And we are just really getting started :) There will be more to come as the PE's come up for cloning. More to follow...

-feds

*70% Isopropyl is used as it takes longer for it to dry than a higher percentage, increasing the window of time to work.

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#6 CoyoteMesc

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Posted 13 March 2008 - 11:31 AM

grow baby grow :D

Good luck feds :thumbup:

#7 prism

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Posted 13 March 2008 - 11:31 AM

*Sits on ground Indian Style and passes Peace pipe to feds....:bow::bow::bow:

#8 joystik

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Posted 13 March 2008 - 12:10 PM

what kind of needle is that?

#9 gsmith1981

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Posted 13 March 2008 - 12:22 PM

:eusa_clap
glad that coring is working out for ya. :thumbup:

#10 Hippie3

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Posted 13 March 2008 - 12:25 PM

:cool:
nice work

#11 fedshtkpndrk

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Posted 13 March 2008 - 01:05 PM

A big thanks to you all, passes the pipe to the left; Thanks Prism :D

what kind of needle is that?


Its a 16ga Joy :)

-feds

#12 suckerfree

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Posted 13 March 2008 - 01:56 PM

nice :eusa_clap

#13 eyeslikedonuts

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Posted 13 March 2008 - 02:17 PM

*plops his beanbag down* simply amazing. I am definately going to give this a try when my fruits are clonable. thanks feds and livewire. this is exactly why I love this site!!!!! :loveeyes: :headbang: . peace, e :teeth:

#14 dta08

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Posted 13 March 2008 - 02:29 PM

So did u inject the core through the ports in the lid of the jar?

#15 Cap'n Murphy

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Posted 13 March 2008 - 02:55 PM

Could a H2O2 be used to clean the biopsy site instead of alcohol?

#16 fedshtkpndrk

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Posted 13 March 2008 - 08:25 PM

So did u inject the core through the ports in the lid of the jar?


:thumbup: exactly


Could a H2O2 be used to clean the biopsy site instead of alcohol?


I have used H202 without success, but I have been pondering using provodine-iodine :) Isopropyl is strong so essentially you are looking to kill the outer tissue and give yourself a safety net of time to transfer the core onto the agar while its in the sterile needle. It also helps a great deal if you draw back the plunger in a sterile environment before performing the biopsy

Please note that this procedure is really only good for cloning, Isolations and transfers on agar should still be done in a GB or in front of a flowhood :)

-feds
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#17 Cap'n Murphy

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Posted 13 March 2008 - 09:41 PM

Comprende

#18 fedshtkpndrk

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Posted 18 March 2008 - 05:02 PM

Some Pics of the PFA Clone jars, bouncing back nicely :) Should be about a week till full colonization of the agar. At that time, we will be making a slurry with the agar and sterile distilled water. Both jars are also contam free :amazed:


:dance:

-feds

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#19 abbr.

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Posted 18 March 2008 - 05:44 PM

Very nice feds, Im pulling up a seat.
Actually, I wish I would have saw this thread
about 3 days ago, lol.
Would have been easier than fumbling around
with an exacto, trying to pull clean tissue from
a nice Goliath cluster.
Last I checked things were'nt looking so good.
I also tried cloning a pin to agar, not looking good either. lol
Maybe I'll try this with some second flush fruits.
:kewl:

#20 Dank Side Of The Shroom

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Posted 19 May 2008 - 09:55 AM

nice work bro. :bow:




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