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OfflinePTreeDish
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Registered: 04/22/18
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Last seen: 3 months, 15 days
PCR & DNA Barcoding
    #26953152 - 09/24/20 05:43 PM (3 years, 4 months ago)

I recently completed my first successful PCR of the ITS genes for fungal DNA barcoding using a combination of Alan Rockefeller and Sigrid Jakob's protocols.

My samples just went out to the lab and I'm expected them back this coming week. It's been a lot of work in getting to this point, not coming from a bio background, but it's incredibly rewarding. The samples include this unidentified mold(?), P. stipticus and P. cinctulus + a few others from my collection.

If you have any questions, I'd be happy to answer them!

Follow me for more fungi and synbio goodness: https://www.instagram.com/everymanbio/



Timelapse of gel electrophoresis results:


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Offlinesporecap
Shedding...

Registered: 07/30/18
Posts: 413
Loc: Europe
Last seen: 1 year, 10 months
Re: PCR & DNA Barcoding [Re: PTreeDish]
    #26953735 - 09/25/20 12:44 AM (3 years, 4 months ago)

:popcorn:
Your insta looks pretty cool as well!


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OfflinePTreeDish
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Registered: 04/22/18
Posts: 353
Last seen: 3 months, 15 days
Re: PCR & DNA Barcoding [Re: sporecap]
    #26954389 - 09/25/20 12:01 PM (3 years, 4 months ago)

Thank you!


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InvisibleJawn876
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Registered: 09/12/18
Posts: 77
Re: PCR & DNA Barcoding [Re: PTreeDish]
    #26981672 - 10/12/20 02:36 PM (3 years, 3 months ago)

Any updates? Im trying to do another run soon and Im wondering where to send it. I don't have a lot of extra money right now so I would really like to make it count. Did you try sending samples out to Genewiz? Or any better luck using MClab? Im unclear if you actually need an institutional affiliation for Genewiz, I made an account there with no questions asked but never sent anything out. Did you try the running a gel lane with a primer? I checked out your interview with Sigrid Jakob, super cool man keep it up!


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OfflinePTreeDish
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Registered: 04/22/18
Posts: 353
Last seen: 3 months, 15 days
Re: PCR & DNA Barcoding [Re: Jawn876]
    #26984304 - 10/13/20 11:34 PM (3 years, 3 months ago)

I just got my custom primers back from Genewiz and diluted them so I should be ready to do another run in the coming week. I'm using Genewiz this time around and will be including a primer and possibly another fruit-body as controls. You don't need an institution with Genewiz but they do need a "Company Name" associated with your account in their database. You can put whatever you want for that on the form. Their chat support is really good too, even helping me with some specifics to my protocol. Glad you checked out the podcast! Appreciate the support. :thumbup:


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OfflinePTreeDish
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Registered: 04/22/18
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Re: PCR & DNA Barcoding [Re: Jawn876]
    #27006966 - 10/27/20 06:59 PM (3 years, 3 months ago)

Took me two tries, but the 2nd time was a charm. Most of samples came back with solid sequence data and great looking chromatograms. :thumbup:



The sequence and chromatogram data are actual. This mold hopped off a P. cinctulus wild print and was previously posted for ID (unsucessfully) here:
https://www.shroomery.org/forums/showflat.php/Number/26752715

Happy to answer any questions.


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InvisibleAndyHinton


Registered: 12/05/16
Posts: 434
Re: PCR & DNA Barcoding [Re: PTreeDish]
    #27017508 - 11/02/20 01:15 PM (3 years, 2 months ago)

Beautiful gel bands, Josh! Did you cut them out and clean them up before sequencing, or did your service let you send in unrefined PCR product?


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OfflinePTreeDish
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Registered: 04/22/18
Posts: 353
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Re: PCR & DNA Barcoding [Re: AndyHinton]
    #27018418 - 11/02/20 10:02 PM (3 years, 2 months ago)

Thanks! Nah, I let the lab do it. It's pretty cheap and seems worth it for the risk mitigation. Do you?


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InvisibleAndyHinton


Registered: 12/05/16
Posts: 434
Re: PCR & DNA Barcoding [Re: PTreeDish]
    #27018808 - 11/03/20 07:30 AM (3 years, 2 months ago)

I had tested an agarose purification kit we had lying around and the Qubit couldn't pick up any DNA in the results. But the problem may also have been a faulty PCR run full of polymer dimers, as the bands weren't so crisp.

After a new PCR run with high-fidelity enzymes, I'll probably not bother to clean it up myself. MGH DNA Core doesn't seem to require it as long as the concentration is correct.


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Offlinetrippleblack
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Registered: 12/01/19
Posts: 355
Last seen: 1 day, 2 hours
Re: PCR & DNA Barcoding [Re: AndyHinton]
    #27019808 - 11/03/20 04:27 PM (3 years, 2 months ago)

what machine are you running cycles with?

any recommendations? i was leaning towards minipcr 8 or 16..


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OfflinePTreeDish
Male


Registered: 04/22/18
Posts: 353
Last seen: 3 months, 15 days
Re: PCR & DNA Barcoding [Re: AndyHinton]
    #27019851 - 11/03/20 04:51 PM (3 years, 2 months ago)

Yea, me and some other folks all kind of agree it's worth the ~$2-4 per sample to have the lab do it for you.


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OfflinePTreeDish
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Registered: 04/22/18
Posts: 353
Last seen: 3 months, 15 days
Re: PCR & DNA Barcoding [Re: trippleblack]
    #27019853 - 11/03/20 04:52 PM (3 years, 2 months ago)

The minipcr is great! I bought an Applied Biosystems GeneAmp PCR 9700 on eBay for about $150 plus $150 for shipping.


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