Fish gDNA isolation
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scoc...@gmail.com
Sep 29, 2014, 3:45:19 PM9/29/14
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Hey guys and gals,
Anyone have experience working with DIY fish genomic DNA extraction? I've been browsing some articles and trying to get an idea of the general recipe but I trust the collective intelligence of The List more than an article when it comes to hands on practical stuff anyway. I'm trying to do some barcoding but I seldom leave the plant kingdom so any advice would be greatly appreciated. Only rules are no columns and no hazards like phenol chloroform. I know its gonna take a bunch of proteinase K but does anyone have a specific go-to animal tissue protocol on hand that they can personally vouch for? Thanks in advance!
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
Anyone have experience working with DIY fish genomic DNA extraction? I've been browsing some articles and trying to get an idea of the general recipe but I trust the collective intelligence of The List more than an article when it comes to hands on practical stuff anyway. I'm trying to do some barcoding but I seldom leave the plant kingdom so any advice would be greatly appreciated. Only rules are no columns and no hazards like phenol chloroform. I know its gonna take a bunch of proteinase K but does anyone have a specific go-to animal tissue protocol on hand that they can personally vouch for? Thanks in advance!
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
SC
Sep 29, 2014, 5:00:45 PM9/29/14
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Curiously, why no columns? You get a nice clean product from a column.
scoc...@gmail.com
Sep 29, 2014, 5:05:02 PM9/29/14
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I meant no column kits. It's for a workshop im running and we have genomic extraction kits but it would be great if there is a quick and dirty method that can give PCRable DNA. I have use a mod of Edwards buffer and cold isopropyl for plants. Curious if there is a shortcut (with drawbacks) for animal tissue (fish different?). Thanks!
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: 'SC' via DIYbio
Sent: 9/29/2014 5:00 PM
To: diy...@googlegroups.com
Subject: [DIYbio] Re: Fish gDNA isolation
Curiously, why no columns? You get a nice clean product from a column.
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Yuriy Fazylov
Sep 30, 2014, 9:54:31 AM9/30/14
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Try Sambrook et al (1989) I haven't read into it personally.
This study modified it for fish DNA fingerprinting http://www.nature.com/hdy/journal/v76/n4/abs/hdy199649a.html
Trying to extract GFP from glow fish?
This study modified it for fish DNA fingerprinting http://www.nature.com/hdy/journal/v76/n4/abs/hdy199649a.html
Trying to extract GFP from glow fish?
scoc...@gmail.com
Sep 30, 2014, 1:11:05 PM9/30/14
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Thanks for the link. No, its not for glowfish gfp. It's for a workshop Im going to run and would rather not blow through my tissue gdna kit if its not needed. We are barcoding fish found in markets across the NYC and matching the claimed species with the COI gene variation as a fingerprint. Basically showing how pcr can help spot fish fraud. Classic lab exercise. Nothing too exciting.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: Yuriy Fazylov
Sent: 9/30/2014 9:54 AM
To: diy...@googlegroups.com
Subject: RE: [DIYbio] Re: Fish gDNA isolation
Try Sambrook et al (1989) I haven't read into it personally.
This study modified it for fish DNA fingerprinting http://www.nature.com/hdy/journal/v76/n4/abs/hdy199649a.html
Trying to extract GFP from glow fish?
This study modified it for fish DNA fingerprinting http://www.nature.com/hdy/journal/v76/n4/abs/hdy199649a.html
Trying to extract GFP from glow fish?
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Cory Tobin
Sep 30, 2014, 1:51:10 PM9/30/14
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The columns are just a convenient way to pass your sample over
diatomaceous earth. Get some diatomaceous earth powder and wash it
with your DNA binding buffer. Then add it you your sample and mix it,
then centrifuge to pellet the powder and pipette off the supernatant.
It's less fool proof since you can potentially suck up the sample, but
way cheaper.
You'll still need all of the same solutions but those you can often
make yourself for pretty cheap, if you can find the recipes. A lot of
the Qiagen recipes are on the interwebs.
I should mention, I've never tried this with genomic DNA. I always
use it for minipreps and it works just as well as the columns
-cory
diatomaceous earth. Get some diatomaceous earth powder and wash it
with your DNA binding buffer. Then add it you your sample and mix it,
then centrifuge to pellet the powder and pipette off the supernatant.
It's less fool proof since you can potentially suck up the sample, but
way cheaper.
You'll still need all of the same solutions but those you can often
make yourself for pretty cheap, if you can find the recipes. A lot of
the Qiagen recipes are on the interwebs.
I should mention, I've never tried this with genomic DNA. I always
use it for minipreps and it works just as well as the columns
-cory
Cathal (Phone)
Sep 30, 2014, 6:54:29 PM9/30/14
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Would love to see the protocols in that paper but..paywall. :(
On 30 September 2014 18:11:01 GMT+01:00, scoc...@gmail.com wrote:
Thanks for the link. No, its not for glowfish gfp. It's for a workshop Im going to run and would rather not blow through my tissue gdna kit if its not needed. We are barcoding fish found in markets across the NYC and matching the claimed species with the COI gene variation as a fingerprint. Basically showing how pcr can help spot fish fraud. Classic lab exercise. Nothing too exciting.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&DTry Sambrook et al (1989) I haven't read into it personally.
From: Yuriy Fazylov
Sent: 9/30/2014 9:54 AM
To: diy...@googlegroups.com
Subject: RE: [DIYbio] Re: Fish gDNA isolation
This study modified it for fish DNA fingerprinting http://www.nature.com/hdy/journal/v76/n4/abs/hdy199649a.html
Trying to extract GFP from glow fish?
--
Sent from my Android device with K-9 Mail. Please excuse my brevity.
Nathan McCorkle
Sep 30, 2014, 7:08:16 PM9/30/14
to diybio
On Tue, Sep 30, 2014 at 3:50 PM, Cathal (Phone) <cathal...@cathalgarvey.me> wrote:
Would love to see the protocols in that paper but..paywall. :(
Yuriy Fazylov
Sep 30, 2014, 8:28:22 PM9/30/14
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Cool. I didn't expect the link to be spot on.
If it is a common food stock like Tilapia. Know that some of those are not true species but hybrid of two species. I think it is done to make them hardier and bigger.
scoc...@gmail.com
Sep 30, 2014, 8:52:39 PM9/30/14
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You learn something new everyday! I didn't know tilapia was bred like that. Thanks for the factoid. I was leaning more towards tuna and salmon since Atlantic and pacific have varied prices.
Does anyone think that Edwards Buffer (Tris, EDTA, SDS, NaCl) plus proteinase K would work as a digest buffer? I use it all the time for plant gdna extraction (minus Prot K) and I just grind in buffer, spin down debris, mix supernatant with cold isoprop, and spin for 10mins. Never had a bad pcr using that method so I'm curious if the buffer is versatile enough to use in animal tissue. I know proteinase K is very robust across detergents, pH, etc. Any thoughts?
Does anyone think that Edwards Buffer (Tris, EDTA, SDS, NaCl) plus proteinase K would work as a digest buffer? I use it all the time for plant gdna extraction (minus Prot K) and I just grind in buffer, spin down debris, mix supernatant with cold isoprop, and spin for 10mins. Never had a bad pcr using that method so I'm curious if the buffer is versatile enough to use in animal tissue. I know proteinase K is very robust across detergents, pH, etc. Any thoughts?
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: Yuriy Fazylov
Sent: 9/30/2014 8:28 PM
To: diy...@googlegroups.com
Subject: Re: [DIYbio] Re: Fish gDNA isolation
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/1f452a39-93a6-4daa-bd3a-e92fdf4db6d2%40googlegroups.com.
Yuriy Fazylov
Sep 30, 2014, 9:26:28 PM9/30/14
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http://primerdigital.com/dna.html (bottom of the site)
scoc...@gmail.com
Sep 30, 2014, 9:30:35 PM9/30/14
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Perfect! Thanks! I'll post results of the workshop to the list if it works well...in case anyone is interested.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/1f1c40d6-a972-4b92-819d-3147bc50d365%40googlegroups.com.
Yuriy Fazylov
Sep 30, 2014, 9:44:48 PM9/30/14
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I am, if only for literature sake.
I'd like to bar-code environmental DNA (eDNA) in a local pond but I hear that qPCR is better at it than regular one. Presently, i don't have access to that equipment or know-how.
Cathal Garvey
Oct 1, 2014, 4:15:40 AM10/1/14
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:D
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Twitter: @onetruecathal, @formabiolabs
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