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InvisibleTeemo 6T3
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Agar cloning question
    #22187207 - 09/04/15 10:12 AM (8 years, 4 months ago)

I have searched around shroomery before posting this question, but i haven't found a straight answer..  :strokebeard2:

So i want know if say i took a clone tissue and grew it out on agar...and i want to make a clone not an isolate out of it, so i know i must transfer sectors to make a clean culture to continue.... So my question is wouldn't transferring that clean section have a chance to be a genetically non fruiting semi-isolate?  (meaning as in its not a fully isolated mono culture)

so wouldn't it be better to just sector all the clean sections (mostly going to be the outer leading parts of the agar tray) and just mix them together in an LC or agar so that it could have an increase chance of maintaining all the genetic variation of the clone i took?

And also one of my past clone experiments was recovering very nicely until it got just infested with Rhizopus stolonifer, so in these cases should i just throw them out since these types of contaminates spread really fast?


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OfflineGrim767
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Re: Agar cloning question [Re: Teemo 6T3]
    #22187227 - 09/04/15 10:17 AM (8 years, 4 months ago)



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OfflinePsilosopherr
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Re: Agar cloning question [Re: Grim767]
    #22187338 - 09/04/15 10:37 AM (8 years, 4 months ago)

People say that the different sets of genetic will cooperate together, and some seem to cooperate better than others. Some say 'conglomerate clones' can be better than isolates but I haven't seen much evidence of this.

Selectively breed some cooperative genetic conglomerates and test it outtt man.


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Offlinekosmokratorshaman
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Re: Agar cloning question [Re: Psilosopherr]
    #22189823 - 09/04/15 07:40 PM (8 years, 4 months ago)

are you asking if you should take a wedge from the edge of the agar tray, as opposed to the center so that you don't accidentally isolate a mono-spore?


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InvisibleToadstool5
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Re: Agar cloning question [Re: kosmokratorshaman]
    #22190989 - 09/04/15 11:45 PM (8 years, 4 months ago)

Quote:

so wouldn't it be better to just sector all the clean sections (mostly going to be the outer leading parts of the agar tray) and just mix them together in an LC or agar so that it could have an increase chance of maintaining all the genetic variation of the clone i took?




That would be your best bet, or drop a wedge from the tip of each sector into a master grain jar together and G2G from there.

It's a balancing act really. If you clone closer to the center of the radial growth the strands will be tangled and you will get a wider array of genetics but you are cloning older cells so it will senesce faster than the hyphal tips would.

To preserve the different genetics without culturing old growth mixing the tips of the sectors in a master is the best option in my opinion.

Quote:

And also one of my past clone experiments was recovering very nicely until it got just infested with Rhizopus stolonifer, so in these cases should i just throw them out since these types of contaminates spread really fast?




Do you feel lucky? :ruggedwink:


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InvisibleTeemo 6T3
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Re: Agar cloning question [Re: Toadstool5]
    #22191323 - 09/05/15 03:07 AM (8 years, 4 months ago)

Quote:

rbalzer said:
People say that the different sets of genetic will cooperate together, and some seem to cooperate better than others. Some say 'conglomerate clones' can be better than isolates but I haven't seen much evidence of this..




Interesting... seems like that would be my best choice then..

Quote:

kosmokratorshaman said:
are you asking if you should take a wedge from the edge of the agar tray, as opposed to the centre so that you don't accidentally isolate a mono-spore?




No, what i meant was instead of taking 1 small wedge from the outer part of the agar tray which could have a certain mixed type of genetics... why not just take all the outer parts by cutting a circular shape (leaving behind the inner mycelium ofc) and transferring it to a clean plate where you can cut it up more into smaller pieces and just mix that up into a grain jar or LC.


Quote:

Toadstool5 said:

That would be your best bet, or drop a wedge from the tip of each sector into a master grain jar together and G2G from there.

It's a balancing act really. If you clone closer to the center of the radial growth the strands will be tangled and you will get a wider array of genetics but you are cloning older cells so it will senesce faster than the hyphal tips would.

To preserve the different genetics without culturing old growth mixing the tips of the sectors in a master is the best option in my opinion.




Thats exactly what i had in mind man!  :highfive1:


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InvisibleToadstool5
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Re: Agar cloning question [Re: Teemo 6T3]
    #22192290 - 09/05/15 11:40 AM (8 years, 4 months ago)

:goodluck:

I'm trying this with a clone i have too! I always took two good looking sectors but why not try 3 or 4? You do have a better chance for contams but it's much better for keeping the symbiosis between different morphologies going strong. :thumbup:


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OfflineMMG
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Re: Agar cloning question [Re: Toadstool5]
    #22255719 - 09/18/15 11:00 AM (8 years, 4 months ago)

What'd you end up doing with the one with Rhizopus stolonifer? I did my first agar dishes and all of my cultures ended up gettting this mold, sucks big time. Is tossing them the best way to go? I want to isolate a clean patch and try growing them again since these are my last culture spores


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InvisibleTeemo 6T3
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Re: Agar cloning question [Re: MMG]
    #22255865 - 09/18/15 11:44 AM (8 years, 4 months ago)

I tossed mine straight away, those buggers just spread so fast you cant even find a place to sector out a clean culture.  :undecided:

I noticed a little wispy white thread jumping over the agar first, ignored it because i thought it must of been my white cat's hair that some how got in there before sterilisation..  and within a day or so it just spread like wild fire.

I'd say the only way you could save the culture is if you monitor your agar and see if it shows the sign of that contaminant (which would look like a piece of tiny white hair jumping over the agar - this is why i tend to use coloured agar because it easier to see it-) and sector the clean part ASAP before it gets infested.

Edit: Why are you starting spores with agar, you're better off just inoculating it to grain > G2G > bulk > fruit > clone tissue > agar

it would be much faster for you to isolate it than starting with spores on agar (unless you're lucky), and you would have a higher percentage of it being a good fruiter, less of a hassle really..


Edited by Teemo 6T3 (09/18/15 12:00 PM)


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OfflineMMG
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Re: Agar cloning question [Re: Teemo 6T3]
    #22256357 - 09/18/15 01:58 PM (8 years, 4 months ago)

Ah man, I'm gonna get some transfers done right now. Maybe I can salvage some clean culture out of them... Here are some pics to illustrate


Those two are galindoi spores which I got off a print, I also did some AA+ and cams. The reason I did agar was because of the small amount of spores I had left, I would then do agar wedges and transfer onto grains, so long for those plans.
Anyway, thanks for your input!
Edit: when I did this plates I knew I was risking my last spores so I did two different agar recipes (potato broth with a bit of honey, and potato broth with a bit of starch flour), weird thing is that all my plates grew this mold, makes me wonder what went wrong so I can fix it next time.
Edit 2: I did do some to grain at first, those failed because of mistakes on the grain prep (that left me with little spores left on the syringes 2 cc <), the 3rd time around I got the grains right (mostly) and my Mexicanas grew well, the AA+'s never colonized the rye grass I put them in (should've done rye berry). But what gives, I'm learning as I go


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Edited by MMG (09/18/15 02:10 PM)


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Invisiblemicro
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Re: Agar cloning question [Re: MMG]
    #22256648 - 09/18/15 03:10 PM (8 years, 4 months ago)

Hmm... I think what we need is something like a benomyl (or other fungicide) resistant strain, so you can add that to the media in addition to bacteriostatics. You might be able to take a very small sector in a still air environment but yeah -- I've met rhizopus and he's an ass xD

That's the big problem with things like this; the contaminants grow many orders of magnitude faster than the mycelium you want. Mushrooms have a niche and are more specialized than your common "swoop in and take over everything" competitors. With a nutritious media like that you're much more likely to get the latter if it's around.

I can't imagine it being that difficult to clone a resistance gene in but I don't know... I only worked with stuff like e. Coli. Not sure if you can just clone a plasmid into the hyphae.


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InvisibleTeemo 6T3
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Re: Agar cloning question [Re: MMG]
    #22256925 - 09/18/15 04:23 PM (8 years, 4 months ago)

Quote:

micro said:
I think what we need is something like a benomyl (or other fungicide) resistant strain, so you can add that to the media in addition to bacteriostatics.




I would of also suggested antibiotics, but i remembered that it will only help with bacteria lol...

Honestly i had a problem with Rhizopus only once, i think it was because i wasn't following my usual sterile procedure, just be sure to be extra sterile next time, because the spores that this fungus produces is just so abundant in the air around us.


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Offlineconfuzzed
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Re: Agar cloning question [Re: micro]
    #22258188 - 09/18/15 09:14 PM (8 years, 4 months ago)

Quote:

micro said:
Hmm... I think what we need is something like a benomyl (or other fungicide) resistant strain, so you can add that to the media in addition to bacteriostatics.




Do this and start your own company!

Call it "microsanto" :lol:


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Invisiblemicro
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Re: Agar cloning question [Re: confuzzed]
    #22258650 - 09/18/15 11:57 PM (8 years, 4 months ago)

maybe if i get this bioinformatics gig

tbh, they have kits for that. you could probably do it at home with some creativity

i've thought of this, for example you could do a PCR if you had the polymerase, restriction digests and primers; just have 5 water baths at different temperatures


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OfflineMMG
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Re: Agar cloning question [Re: micro]
    #22259777 - 09/19/15 11:28 AM (8 years, 4 months ago)

I used a SAB for the agar dishes which is why I find it strange that they contamed.
Do you guys PC your syringes prior to use?
I'm thinking I'll try this as a measure to avoid contams this next time around.
What media do you guys usually use for your dishes?


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Invisiblemicro
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Re: Agar cloning question [Re: MMG]
    #22259834 - 09/19/15 11:44 AM (8 years, 4 months ago)

If they are empty glass syringes...

Not ones with spores in them of course.


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InvisibleToadstool5
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Re: Agar cloning question [Re: MMG]
    #22260034 - 09/19/15 12:29 PM (8 years, 4 months ago)

I boil the water for my syringes for 20min, suck up and expel a full syringe worth of water three times, fill and cap the syringe, put it in a plastic bag and let cool before using to make a print.

PDYA or MEA for me in PP5 containers, no pour tek in a SAB. I occasionally get a bacteria colony but if my inoculate or culture is clean they always come out clean. If I use syringes on agar I use a small ~24gauge needle with a 3ml syringe and make the spore solution dark. With slightly firm agar and concentrated but small drops i dont have issues doing spore water->agar. Less concentrated spore water gives me issues sometimes with blanks but still works with the small needles.

Do you HEPA filter your air before letting it settle? Do you disinfect your work station and gloves? Do you wear tyvek sleeves and a surgical mask? Flame sterilizing? Are your petri's arriving sterile if you ship?

Go through your procedure very carefully and think of any possible vectors as you work, then modify your technique on the next round and see how or if it improves. Normally its something simple and small that you forget to do.


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Invisiblemicro
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Re: Agar cloning question [Re: Toadstool5]
    #22260061 - 09/19/15 12:36 PM (8 years, 4 months ago)

You might want to look into bottles with septum caps,,, Makes life easier IMO

Just sterilize some water in them (with the caps a bit loose) then store on the shelf.


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InvisibleToadstool5
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Re: Agar cloning question [Re: micro]
    #22260131 - 09/19/15 12:53 PM (8 years, 4 months ago)

It's easy enough to boil the water in the syringes or use a 1/2pt jar with an RTV silicon port, I'm also a VERY cheap bastard!

Septum bottles do rock if you have them and the seal is way better than a mason jar :lol:


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OfflineMMG
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Re: Agar cloning question [Re: Toadstool5]
    #22263074 - 09/20/15 12:54 AM (8 years, 4 months ago)

I'm gonna order some standardized media soon, see how it compares to boiled potatoes.
Sterlization is pretty basic.
What I do is spray some soapy water with air freshener on my SAB to settle the air in there.
I use gloves and sleeves up to my elbow, soaked with alcohol and my syringe was sterilized between inoculations this time.
I don't treat my air with any filters, but my local CL has some pretty cheap air filters for sale, I might pick one up soon
Thanks for the tips!

As far as the agar it might've been that my syringes were not too concentrated, they where pretty diluted so that could've been a partial catalyst.
Does having extra water in there harm the colonization process? more chance for contams to be in there?


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