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OfflinecronicrFacebook
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Re: Anyone Tried Cloning This Way??? [Re: Peteyboy] * 2
    #23916803 - 12/11/16 04:11 PM (7 years, 1 month ago)

Pins are great because they are aggressive n u can just drop them on a plate


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Re: Anyone Tried Cloning This Way??? [Re: Peteyboy] * 1
    #23916813 - 12/11/16 04:13 PM (7 years, 1 month ago)

Quote:

KauaiOrca said:

How does mold enter the equation?




Bacteria is a vector for mold.


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Re: Anyone Tried Cloning This Way??? [Re: Pastywhyte]
    #23916837 - 12/11/16 04:18 PM (7 years, 1 month ago)

Quote:

Pastywhyte said:
Quote:

KauaiOrca said:

How does mold enter the equation?




Bacteria is a vector for mold.




OK, so let's say I cut the stem at the base  with at least an inch or so solid stem at the bottom.  Then I dip the bottom of the stem in alcohol.  Then I wipe it with a lysol or chlorox wipe.  Then I take a red hot knife and cut a 1/4-1/2 slice off the end and then I poke the red hot needle through the bottom of the stem (been dipped i alcohol, wiped with chlorox and cut with a red hot knife)  Do you still think bacteria and mold are likely to play a role here?


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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Re: Anyone Tried Cloning This Way??? [Re: KauaiOrca]
    #23916855 - 12/11/16 04:22 PM (7 years, 1 month ago)

Quote:

KauaiOrca said:
Quote:

Pastywhyte said:
Quote:

KauaiOrca said:

How does mold enter the equation?




Bacteria is a vector for mold.




OK, so let's say I cut the stem at the base  with at least an inch or so solid stem at the bottom.  Then I dip the bottom of the stem in alcohol.  Then I wipe it with a lysol or chlorox wipe.  Then I take a red hot knife and cut a 1/4-1/2 slice off the end and then I poke the red hot needle through the bottom of the stem (been dipped i alcohol, wiped with chlorox and cut with a red hot knife)  Do you still think bacteria and mold are likely to play a role here?




I think you just chemically fucked up your sample.


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Re: Anyone Tried Cloning This Way??? [Re: KauaiOrca] * 3
    #23916861 - 12/11/16 04:23 PM (7 years, 1 month ago)

Dude you are missing the piunt so badly here...it is simply not a good plan and on many levels.
Would be faster to clone it properly then to trie cut corners so you can jump to rye in a haste...this hobby is one you don't rush.


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Re: Anyone Tried Cloning This Way??? [Re: cronicr]
    #23916884 - 12/11/16 04:29 PM (7 years, 1 month ago)

Quote:

cronicr said:
Dude you are missing the piunt so badly here...it is simply not a good plan and on many levels.
Would be faster to clone it properly then to trie cut corners so you can jump to rye in a haste...this hobby is one you don't rush.




Actually, I'm in no rush at all.  Have several really good LC's that work perfectly with my system, so it's not about rushing ... And it's not about cutting corners either. 

I've had just as good of results starting spores on a small jar (pastyplate size) of RGS as on Agar ... Have also pulled invitro pins and put them on RGS too and worked fine ...  I've just never tried cloning a mature or grown past a pin sized fruit and was thinking about it that's all.


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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Re: Anyone Tried Cloning This Way??? [Re: KauaiOrca] * 2
    #23916906 - 12/11/16 04:35 PM (7 years, 1 month ago)

The two spots to clone from are the base and the cap where the myc is rapidly expanding from...the middle is mostly cell enlargement


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Re: Anyone Tried Cloning This Way??? [Re: cronicr]
    #23916934 - 12/11/16 04:43 PM (7 years, 1 month ago)

Yea you can't clean it with the needle method. Only did it five or six times with a lc of trich half the time. Have fun but if you have clean lcs just spent the time on agar with that fruit. Also spores to grain can be just as fast or much slower. I've done jars 100% form spores in 14 days. Or no growth ever.


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Re: Anyone Tried Cloning This Way??? [Re: tump] * 1
    #23917392 - 12/11/16 06:37 PM (7 years, 1 month ago)

My man....you got two of the best guys in the business here giving you advice...TAKE IT, you should feel privileged they are taking the time to help you...:shrug:


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Re: Anyone Tried Cloning This Way??? [Re: KauaiOrca]
    #23917583 - 12/11/16 07:15 PM (7 years, 1 month ago)

Quote:

KauaiOrca said:
Given that mushroom stems are generally fairly hollow, I had an idea in terms of cloning that would be an easy, one-step process.  I would take a sterile syringe with about 3-4 ML's of water and then but the mushroom near the base with a red hot knife,  At the base, the stem is not hollow so through the solid, meaty bottom, the needle goes up and into hollow part of the stem, always staying inside the stem ... I then inject the sterile water into the hollow stem, leaving the needle in there ... shake it a bit an then suck the water back into the needle, which never left the stem ... take the needle out, heat it quickly and then inject the water into a small jar of RGS. 

I tried the poke the stem with the needle and try to get a piece of it technique that never worked for me, but am wondering if anyone's done this?  Theoretically, it's sterile in the hollow part of the mushroom, right?  I'd think the water injected would pick up enough cells to get a small jar of RGS going which could then be slurried for liquid innoculant.  Could also, I guess do it to agar too.


Any thoughts?



sounds like the idea someone who is trying to avoid doing things the right way comes up with. eats might like this method


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Re: Anyone Tried Cloning This Way??? [Re: bodhisatta]
    #23917690 - 12/11/16 07:42 PM (7 years, 1 month ago)

Quote:

bodhisatta said:
Quote:

KauaiOrca said:
Given that mushroom stems are generally fairly hollow, I had an idea in terms of cloning that would be an easy, one-step process.  I would take a sterile syringe with about 3-4 ML's of water and then but the mushroom near the base with a red hot knife,  At the base, the stem is not hollow so through the solid, meaty bottom, the needle goes up and into hollow part of the stem, always staying inside the stem ... I then inject the sterile water into the hollow stem, leaving the needle in there ... shake it a bit an then suck the water back into the needle, which never left the stem ... take the needle out, heat it quickly and then inject the water into a small jar of RGS. 

I tried the poke the stem with the needle and try to get a piece of it technique that never worked for me, but am wondering if anyone's done this?  Theoretically, it's sterile in the hollow part of the mushroom, right?  I'd think the water injected would pick up enough cells to get a small jar of RGS going which could then be slurried for liquid innoculant.  Could also, I guess do it to agar too.


Any thoughts?



sounds like the idea someone who is trying to avoid doing things the right way comes up with. eats might like this method




Appreciate all the responses and suggestions!  i have no problem at all doing the pins to agar process and currently have 3 of them in process ... Am not trying to cut time off or anything like that.  Was simply curious if anyone had tried this idea, that's all. 

What I think I'll do is try it both onto RGS and Agar and see what happens.  Total cost of RGS for the experiment is less than 2 cents.  Who knows, maybe it will work.  I like trying new things. 

Thanks again.


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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Re: Anyone Tried Cloning This Way??? [Re: KauaiOrca]
    #23918312 - 12/12/16 12:08 AM (7 years, 1 month ago)

Pictures either way


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Re: Anyone Tried Cloning This Way??? [Re: tump]
    #23924967 - 12/14/16 04:53 AM (7 years, 1 month ago)

Quote:

tump said:
Pictures either way




First attempt update:  I did this technique and squirted 3ml of sterile water into a medium sized fruit through the meaty bottom into the hollow stem, left the needle in... kinda squeezed massaged the meaty bottom hopefully dislodging some of those cells into the mix and then sucked back 1.5ml ... flamed the needle again and quickly squirted that water into a zip loc pint jar with about a half inch of RGS in it.  5 days later a nice half dollar sized clump of "clean looking" growth that I just shook up.  Will update when jar fully colonizes ... intend to do a G2G transfer into 3 quart sized zip locs of RGS and see what happens.  If it grows out, it's an agar-less way to clone direct to grain.  We'll see... so far, no visible contams but too early to tell.

Clone Water Test #1 - Medium sized, tallish fruit 2-3 days from full sized
Day 1: Extracted 1.5ml clone water from inside fruit with syringe - injected to small jar of RGS
Day 5: Quarter sized clean looking growth on small amount of RGS
Day 5: Shook the jar to accelerate colonization
Day 6: After shake, colonizing quickly ... no signs of contamination


Clone Water Test #2 - Early Stage - Stubby Fat Fruit
Day 1: Extracted <1ml clone water from inside fat, stubby fruit with syringe - injected to small jar of RGS
Day 3: First signs of white specks appearing on RGS

Next Step: If no visible contamination - G2G 3 quart zip loc jars (RGS) and try to fruit it. 

Are there other direct clone to grain (no agar step) methods that have ever worked?


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


Edited by KauaiOrca (12/15/16 06:51 AM)


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Re: Anyone Tried Cloning This Way??? [Re: KauaiOrca]
    #23928111 - 12/15/16 12:21 AM (7 years, 1 month ago)

Good point. Ill try this sometime with injecting to some plates and lc jars made pasty style.


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Re: Anyone Tried Cloning This Way??? [Re: tump]
    #23928630 - 12/15/16 08:47 AM (7 years, 1 month ago)

Quote:

tump said:
Good point. Ill try this sometime with injecting to some plates and lc jars made pasty style.




First two attempts are growing nicely, certainly much faster than spores and appear to be contam free although still too early to tell.  If this works, what an easy way to clone different fruits with very low risk of contamination.  And can go straight to a growing medium too to test if the clone produces!    If this works, my need for agar will go down dramatically.  Hardly even need an SAB to do this.


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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Re: Anyone Tried Cloning This Way??? [Re: KauaiOrca]
    #23928661 - 12/15/16 09:02 AM (7 years, 1 month ago)

I might suggest testing on agar. Bacteria can be hard to spot in grains sometimes.


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Re: Anyone Tried Cloning This Way??? [Re: Pastywhyte]
    #23928681 - 12/15/16 09:14 AM (7 years, 1 month ago)

Quote:

Pastywhyte said:
I might suggest testing on agar. Bacteria can be hard to spot in grains sometimes.




That's probably a good thing to do simultaneously.  If 3-4 in a row come out contam free, it's probably an indication that the method is reliable. 

Out of curiosity, wouldn't bacteria show up in the small RGS jars I'm doing?  I mean if those jars produce pins and there's no VISIBLE sign of bacteria, are my chances pretty high that bacteria is not there?  Keep in mind, I have no desire at all to spawn to bulk and/or create dozens and dozens of jars from one small sample.  If anything, I might create an LC from it and those are pretty easy to see if they're thriving/healthy or not.

I'm more thinking for a small contained system ...

Spores to RGS > clone water the good fruits > clone water to small RGS jar invitro > if small RGS jar is clean, G2G to 3-4 muda style jars... and wait for the good fruits ... then rinse and repeat clone water step with the best fruits pretty much indefinitely.

The thing is it's so easy to just pick a nice fruit, flame knife and cut near the bottom, flame a needle and poke up through the bottom and up into the stem, draw out some clone water and inject it into a small jar of RGS ... About 30 seconds and I've got a clone growing on RGS that will tell me if it pins fast.

Appreciate your advice here.


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


Edited by KauaiOrca (12/15/16 09:22 AM)


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Re: Anyone Tried Cloning This Way??? [Re: KauaiOrca]
    #23928718 - 12/15/16 09:32 AM (7 years, 1 month ago)

Bacteria is easier to see on agar than in grain due to agar being a 2D media. I have found through many tests for instance that grain that looks 100% clean often shows bacteria when tested. Put GLC to agar and many times it shows lots of bacteria. However many times that same GLC can colonize grain and even be spawned.

You may be thinking "why worry about the bacteria then if you can still spawn it?" The answer of course is that in low populations bacteria isn't much issue for mycelium. However a small amount of bacteria can cause problems if conditions become more favorable for it. So while 9 times out 10 you might get away with a bit of bacteria in the inoculate, on the 10th go you experience a temp spike for a day, or the moisture content is off just a bit, or you have just a little more spilled starch. Now the bacteria is thriving and while you still might not see or smell it, it's there driving down yield and being a mold vector during spawn runs.

Of course loads of people feel that those are acceptible risks to take. But all it takes is one big expansion and massive project fail to really put someone off the game. Start with guaranteed clean inoculate and your success gets up to 99% and your yields rise. Use dubious inoculate and suddenly your success drops, yields drop, and troubleshooting vectors becomes harder.


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Re: Anyone Tried Cloning This Way??? [Re: Pastywhyte]
    #23928733 - 12/15/16 09:39 AM (7 years, 1 month ago)

Quote:

Pastywhyte said:
Bacteria is easier to see on agar than in grain due to agar being a 2D media. I have found through many tests for instance that grain that looks 100% clean often shows bacteria when tested. Put GLC to agar and many times it shows lots of bacteria. However many times that same GLC can colonize grain and even be spawned.

You may be thinking "why worry about the bacteria then if you can still spawn it?" The answer of course is that in low populations bacteria isn't much issue for mycelium. However a small amount of bacteria can cause problems if conditions become more favorable for it. So while 9 times out 10 you might get away with a bit of bacteria in the inoculate, on the 10th go you experience a temp spike for a day, or the moisture content is off just a bit, or you have just a little more spilled starch. Now the bacteria is thriving and while you still might not see or smell it, it's there driving down yield and being a mold vector during spawn runs.

Of course loads of people feel that those are acceptible risks to take. But all it takes is one big expansion and massive project fail to really put someone off the game. Start with guaranteed clean inoculate and your success gets up to 99% and your yields rise. Use dubious inoculate and suddenly your success drops, yields drop, and troubleshooting vectors becomes harder.




P - theoretically, let's say I do a multispore grow, pick a killer fruit, do the clone water tek and it grows some nice fruits ... if it do clone water with those fruits (Gen 2) am I still working with the same genetics even though the clone water came from a different fruit?  Would this hold true on Gen 3, 4, 5, etc?  Will all the fruits from that first clone water starter produce the same cloning results?  And for how many generations do you think before dropoff is likely?


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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Re: Anyone Tried Cloning This Way??? [Re: KauaiOrca]
    #23928749 - 12/15/16 09:46 AM (7 years, 1 month ago)

There will probably be a degree of genetic shift, impossible to say how much. I wouldn't try t push expansion like that more than 5 times as you are really burning up p values that way. Highly inefficient.


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