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Offlinemyceliation
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New to agar, questions on first clone attempt
    #28000718 - 10/15/22 05:41 PM (2 years, 3 months ago)

I made my first attempt at cloning using malt extract agar with some mixed results. The instructions say to boil the MEA and water for 15 minutes which I did, however at the end it was becoming darker and chunky--it looked better after only ~5 minutes of boiling. I let it cool down somewhat and poured the agar into some Glad mini containers (vent hole made in side near top, two layers of micropore tape over it). I put the lids on and threw it into the PC with some grain jars and did 15psi for 2 hours.

After the PC, I noticed a lot of condensation on the inside, and even some puddling on the agar of some. I checked the agar consistency on one and noticed that it wasn't like jello like I was expecting--it was more like baby food. Flat and shiny on top but a weak consistency overall.

Here's some questions that have arisen on my technique:
-Is there any necessity to boil the mixture for a full 15 minutes, or is just a few minutes enough? It looks like shorter would produce better agar.

-Is there any harm in PC'ing for 2 hours instead of the recommended 15-30 minutes?

-Is there anything else that I can do except for letting the agar cool completely before putting the lid on to avoid condensation? I did not use foil since according to the tek I read, it was unnecessary given the location of the vent hole I made.

-I've got mycelium growth on the other plates and I'm planning on transferring sections to new agar plates to ensure clean cultures. What size squares are typically used for transfers to new plates?

Thanks for helping me get my technique squared away.

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OfflineKROM
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Re: New to agar, questions on first clone attempt [Re: myceliation]
    #28000741 - 10/15/22 05:50 PM (2 years, 3 months ago)

1- 15 minutes boiling agar is waaaaay too long in my experience. Once the solids dissolve, and it feels a bit more viscous, itโ€™s fine.

2- pcing for longer than 30-45 minutes would likely caramelize the sugars, so itโ€™s not a great idea.

3- Iโ€™m not sure I understand. You pc the media bottle with no lid? Or prepoured plates?

4- smaller is better in my opinion.


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Offlinemyceliation
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Re: New to agar, questions on first clone attempt [Re: KROM]
    #28000764 - 10/15/22 06:05 PM (2 years, 3 months ago)

Quote:

KROM said:
3- Iโ€™m not sure I understand. You pc the media bottle with no lid? Or prepoured plates?





Prepoured plates--I boiled the powder and water and poured it into the mini rounds, and then lids on and into the PC. I'm wondering if letting it cool to room temp before putting the lids on and PC'ing is what would eliminate the condensation.

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OfflineExbat
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Re: New to agar, questions on first clone attempt [Re: myceliation]
    #28000769 - 10/15/22 06:08 PM (2 years, 3 months ago)

I believe letting the PC/plates cool down slowly can help avoid condensation, in addition to waiting to put the lids on the plates until after they've cooled.

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OfflineKROM
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Re: New to agar, questions on first clone attempt [Re: Exbat]
    #28000798 - 10/15/22 06:29 PM (2 years, 3 months ago)

Oooh, yeah, that makes sense. I let them cool before putting the lids on, though Iโ€™m not sure it makes much of a difference. I did find that doing this really cut down annoying condensation in my pre-poured plates. Finally, though itโ€™s not a great solution, in a pinch you can carefully pour off any extra water from your plate in an SAB (or laminar flow) before performing transfers.


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Offlinecozmyc
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Re: New to agar, questions on first clone attempt [Re: KROM] * 1
    #28000830 - 10/15/22 06:56 PM (2 years, 3 months ago)

Quote:

KROM said:
1- 15 minutes boiling agar is waaaaay too long in my experience. Once the solids dissolve, and it feels a bit more viscous, itโ€™s fine.

2- pcing for longer than 30-45 minutes would likely caramelize the sugars, so itโ€™s not a great idea.

3- Iโ€™m not sure I understand. You pc the media bottle with no lid? Or prepoured plates?

4- smaller is better in my opinion.



:whathesaid:
I would use AT LEAST 3 layers of pore tape. I do 4.


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Nothing matters so everything does :sunny:

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Offlinemyceliation
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Re: New to agar, questions on first clone attempt [Re: KROM]
    #28000838 - 10/15/22 07:00 PM (2 years, 3 months ago)

Quote:

KROM said:
Oooh, yeah, that makes sense. I let them cool before putting the lids on, though Iโ€™m not sure it makes much of a difference. I did find that doing this really cut down annoying condensation in my pre-poured plates. Finally, though itโ€™s not a great solution, in a pinch you can carefully pour off any extra water from your plate in an SAB (or laminar flow) before performing transfers.




Good stuff, I'll try the method from that link next time.

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Offlinemyceliation
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Re: New to agar, questions on first clone attempt [Re: cozmyc]
    #28000839 - 10/15/22 07:02 PM (2 years, 3 months ago)

Quote:

cozmyc said:
Quote:

KROM said:
1- 15 minutes boiling agar is waaaaay too long in my experience. Once the solids dissolve, and it feels a bit more viscous, itโ€™s fine.

2- pcing for longer than 30-45 minutes would likely caramelize the sugars, so itโ€™s not a great idea.

3- Iโ€™m not sure I understand. You pc the media bottle with no lid? Or prepoured plates?

4- smaller is better in my opinion.



:whathesaid:
I would use AT LEAST 3 layers of pore tape. I do 4.




Is the purpose of adding additional layers of pore tape to reduce the loss of moisture from the plates? Or something else?

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Offlinecozmyc
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Re: New to agar, questions on first clone attempt [Re: myceliation]
    #28000843 - 10/15/22 07:05 PM (2 years, 3 months ago)

Mpore tape actually has huge holes compared to spore size. extra protection. No pours always have some condensation. Iv'e found it clears up after a week or so though.


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You're conscious population 2 stardust
----------------------
Nothing matters so everything does :sunny:

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Offlinemyceliation
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Re: New to agar, questions on first clone attempt [Re: cozmyc]
    #28000971 - 10/15/22 08:31 PM (2 years, 3 months ago)

Very helpful, I'll put this advice into practice. Thanks.

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InvisibleMeanValley
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Re: New to agar, questions on first clone attempt [Re: myceliation]
    #28001126 - 10/15/22 09:55 PM (2 years, 3 months ago)

Size of transfer donโ€™t matter at al. Everyone over thinks it. I take sizes of grains of rice to mini starburst sizes. None of it matters. Just get a clean sample and keep transferring at least 3-4 times. Then wedge it out. Presto. At this rate Iโ€™ll be a TC in a week with this rocket science answers

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OfflineRusty2096
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Re: New to agar, questions on first clone attempt [Re: MeanValley]
    #28001133 - 10/15/22 10:01 PM (2 years, 3 months ago)

Quote:

MeanValley said:
Size of transfer donโ€™t matter at al. Everyone over thinks it. I take sizes of grains of rice to mini starburst sizes. None of it matters. Just get a clean sample and keep transferring at least 3-4 times. Then wedge it out. Presto. At this rate Iโ€™ll be a TC in a week with this rocket science answers




OP, ignore the raging troll he's been doing that in every thread.

Dude - you will never be a TC if you can't grasp the simple fact that the bigger the transfer, the more chances you have or bringing contam with.


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Offlinemyceliation
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Re: New to agar, questions on first clone attempt [Re: Rusty2096]
    #28023714 - 10/29/22 09:46 PM (2 years, 2 months ago)

As I'm to the point of transferring from agar to grain jars, can I get opinions on the ideal number of 1q jars to be inoculated per agar plate?

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Offlinecozmyc
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Re: New to agar, questions on first clone attempt [Re: myceliation]
    #28024591 - 10/30/22 02:34 PM (2 years, 2 months ago)

If you're sure you have a good plate.1/8-1/4 slice per quart. Shake well :mushroom2:


--------------------
You're conscious population 2 stardust
----------------------
Nothing matters so everything does :sunny:

Edited by cozmyc (10/30/22 02:34 PM)

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Offlinemyceliation
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Re: New to agar, questions on first clone attempt [Re: cozmyc]
    #28025312 - 10/30/22 09:45 PM (2 years, 2 months ago)

Quote:

cozmyc said:
If you're sure you have a good plate.1/8-1/4 slice per quart. Shake well :mushroom2:




Awesome thanks.

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