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Gel Solubilization Buffer Composition

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Jagadish Katam

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Jan 9, 2010, 11:40:12 AM1/9/10
to met...@magpie.bio.indiana.edu

Hi,

I am working on purifying the DNA by gel extraction procedure and are using the commercially available kits for this purpose. A yellow color gel solubilization buffer is used to dissolve the sliced gel.

Here i would know the composition of this gel solubilization buffer.

Any suggestions will be greatly appreciated.

Thanks and Best Regards,
Jag


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wattne

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Jan 9, 2010, 1:42:04 PM1/9/10
to
On 9 Jan., 16:40, Jagadish Katam <jagadish...@hotmail.com> wrote:
> Hi,
>
> I am working on purifying the DNA by gel extraction procedure and are using the commercially available kits for this purpose. A yellow color gel solubilization buffer is used to dissolve the sliced gel.
>
> Here i would know the composition of this gel solubilization buffer.
>
> Any suggestions will be greatly appreciated.
>
> Thanks and Best Regards,
> Jag

Hi Jag!
Sounds like the buffer from QIAGEN, right? Why aren't you more
precise?? How should anyone help you if we don't even know what kind
of stuff you are using???
You can bet that the buffer composition is secret because otherwise
QIAGEN couldn't sell their kit anymore; what do you think? Try a
Google, and you will either find out or not. Why should we know the
exact formulation????
Thanks and Best Regards,
watnne

Nick Theodorakis

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Jan 9, 2010, 3:40:27 PM1/9/10
to
On Jan 9, 11:40 am, Jagadish Katam <jagadish...@hotmail.com> wrote:
> Hi,
>
> I am working on purifying the DNA by gel extraction procedure and are using the commercially available kits for this purpose. A yellow color gel solubilization buffer is used to dissolve the sliced gel.
>
> Here i would know the composition of this gel solubilization buffer.
>

In general, agarose gel solubilization can be done with chaotropic
salts such as NaI, sodium perchlorate, or guanidine HCl. These also
work well in conjunction with silica-based binding resins because DNA
binding to silica is promoted in high salt. I doubt the company will
tell you exactly what is in their buffer, but it is likely similar to
one the salts I mentioned, possibly with additives added as a
stabilizer or other use.

Nick

--
Nick Theodorakis
nick_the...@hotmail.com
contact form:
http://theodorakis.net/contact.html


chovek69

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Jan 10, 2010, 4:51:55 AM1/10/10
to
On Jan 9, 10:40 pm, Nick Theodorakis <nick.theodora...@gmail.com>
wrote:
> nick_theodora...@hotmail.com
> contact form:http://theodorakis.net/contact.html

Hi,
As pointed by Nick this is best done by high molar chaotropic salts.
See detailed protocol here:

http://books.google.bg/books?id=Mmne6oC8VhIC&pg=PA123&lpg=PA123&dq=agarose+gel+solubilization&source=bl&ots=JhNritZxi_&sig=1GaJXDGphGOToNAYbrXOHjs4j38&hl=bg&ei=ZpVJS_zBHIb-mQOI6NDXDw&sa=X&oi=book_result&ct=result&resnum=3&ved=0CCAQ6AEwAg#v=onepage&q=agarose%20gel%20solubilization&f=false

Hope this helps
Ivan

Kyle Legate

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Jan 10, 2010, 7:33:55 AM1/10/10
to
Jagadish Katam wrote:
> Hi,
>
> I am working on purifying the DNA by gel extraction procedure and are using the commercially available kits for this purpose. A yellow color gel solubilization buffer is used to dissolve the sliced gel.
>
> Here i would know the composition of this gel solubilization buffer.
>
> Any suggestions will be greatly appreciated.
>
If it's yellow, it's most certainly NaI. Here's the solution I have used
in the past:

90.8 g NaI and 1.5 gm Na2SO3 in 100 ml of water. Filter through 0.45 um
filter and add another 0.5 g Na2SO3. Store in the coldroom in a dark bottle.

Suresh Kumar

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Jan 11, 2010, 4:13:39 PM1/11/10
to met...@oat.bio.indiana.edu

Here is couple of different buffer composition.

Gel solubilization buffer -I

60% Guanidine Thiocyanate (5.36M)
140mM MES (2-[N-Morpholino] ethanesulfonic acid)
0.006% Phenol Red

Use 3 Volume of buffer to 1 gel volume.

Reference: http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0007750

Phenol Red is only a pH indicator. It changes color from yellow to red over the pH range 6.8 to 8.4.

Gel solubilization buffer -II

6M Guanidine Thiocyante
50mM Tris-HCl pH 7.5
20mM EDTA pH 8.0

Use 3 Volume of buffer to 1 gel volume.


Hope this helps.

Suresh

------------------------------

Message: 2
Date: Sat, 9 Jan 2010 22:10:12 +0530
From: Jagadish Katam <jagad...@hotmail.com>
Subject: Gel Solubilization Buffer Composition
To: <met...@magpie.bio.indiana.edu>
Message-ID: <SNT124-W189ABD0BA...@phx.gbl>
Content-Type: text/plain; charset="iso-8859-1"


Hi,

I am working on purifying the DNA by gel extraction procedure and are using the commercially available kits for this purpose. A yellow color gel solubilization buffer is used to dissolve the sliced gel.

Here i would know the composition of this gel solubilization buffer.

Any suggestions will be greatly appreciated.

Thanks and Best Regards,
Jag



_________________________________________________________________
Windows 7: Find the right PC for you. Learn more.
http://windows.microsoft.com/shop

------------------------------

Message: 3
Date: Sat, 9 Jan 2010 12:40:27 -0800 (PST)
From: Nick Theodorakis <nick.the...@gmail.com>
Subject: Re: Gel Solubilization Buffer Composition
To: met...@net.bio.net
Message-ID:
<f87be422-8f3e-4683...@j19g2000yqk.googlegroups.com>
Content-Type: text/plain; charset=ISO-8859-1

On Jan 9, 11:40�am, Jagadish Katam <jagadish...@hotmail.com> wrote:
> Hi,
>
> I am working on purifying the DNA by gel extraction procedure and are using the commercially available kits for this purpose. A yellow color gel solubilization buffer is used to dissolve the sliced gel.
>
> Here i would know the composition of this gel solubilization buffer.
>

In general, agarose gel solubilization can be done with chaotropic


salts such as NaI, sodium perchlorate, or guanidine HCl. These also
work well in conjunction with silica-based binding resins because DNA
binding to silica is promoted in high salt. I doubt the company will
tell you exactly what is in their buffer, but it is likely similar to
one the salts I mentioned, possibly with additives added as a
stabilizer or other use.

Nick

--
Nick Theodorakis


nick_the...@hotmail.com
contact form:
http://theodorakis.net/contact.html


------------------------------

Message: 4
Date: Sat, 9 Jan 2010 18:19:29 -0700 (MST)
From: "Dr. Hiranya S. Roychowdhury" <hroy...@nmsu.edu>
Subject: Re: hi .......help me plz
To: "Amol Ghodke" <amo...@gmail.com>
Cc: met...@magpie.bio.indiana.edu
Message-ID: <1120.71.33.47.214....@webmail.nmsu.edu>
Content-Type: text/plain;charset=iso-8859-1

Do you have access to a library? If you do, perhaps you may check out a
few of the Methods in Enzymology volumes for zymogram protocols. If you
have access to the internet, Google search of the protocols (both zymogram
and protein elution from gels - whether acryl. or starch) will show you
several protocols. Protein/peptide elution from gels is quite simple -
works with electrophoresis or simple diffusion/dialysis methods.


> hi all...
> i have to screen proteinase inhibitors!
> for that i have to do zymography.
> can any one plz give me the detail protocol for that
> and plz tell me how to recover screened protein from gel efficiently
> without
> using highly sofasticated instuments
> plz help me
> thanx
>
> --
> Mr. Amol Bharat Ghodke
> M.Sc. Agril.Biotechnology
> A/P - Kanhur Pathar
> Tal.- Parner,
> Dist.- Ahmednagar
> Pin - 414303
> Mob. 09970865283/09021257663
> _______________________________________________
> Methods mailing list
> Met...@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>


--
Hiranya S. Roychowdhury, Ph.D.
Asst. Professor,
Health & Public Services
Dona Ana Community College
New Mexico State University
Las Cruces, NM 88003

------------------------------

Message: 5
Date: Sun, 10 Jan 2010 11:06:34 +0200
From: Yoram Gerchman <gerc...@research.haifa.ac.il>
Subject: yeast estrogen screen?
To: met...@oat.bio.indiana.edu
Message-ID: <1263114394.4...@webmail.haifa.ac.il>
Content-Type: text/plain; charset=windows-1255


Greetings all
I am trying to get some version of the Yeast Estrogen Screen Assy for some
enviormental work (beta-gal, GFP, ...). Does anyone know were does one get the
yeast and plasmid from?
Many thanks
Yoram

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End of Methods Digest, Vol 56, Issue 8
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angusr...@gmail.com

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Oct 30, 2017, 8:18:17 AM10/30/17
to
Hi Watnne

I found all the other replies to Jag's question helpful and constructive. Yours felt fairly malicious. Please refrain from comments like this in the future, its a waste of time and damages the community feeling of places like this.

Regards

Gus

Hiranya Roychowdhury

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Nov 10, 2017, 12:46:13 PM11/10/17
to DK, met...@magpie.bio.indiana.edu
I had found a simple centrifugal extrusion of the DNA from the gel to be gentler and less costly.


Hiranya S. Roychowdhury, Ph.D.
Professor
Department of Science
Division of Science, Engineering and Math
NMSU-Dona Ana Community College
575 527 7725 (office)

Curriculum & Instruction Committee
Human Anatomy and Physiology Society<http://www.hapsweb.org/>
[http://www.hapsweb.org/graphics/header.jpg]
________________________________
From: methods...@oat.bio.indiana.edu <methods...@oat.bio.indiana.edu> on behalf of DK <d...@no.email.thankstospam.net>
Sent: Thursday, November 9, 2017 5:25:54 PM
To: met...@magpie.bio.indiana.edu
Subject: Re: Gel Solubilization Buffer Composition

I don't know about thje feelings but here is the composition:
5.5 M guanidine thiocyanate, 20 mM Tris-HCl, pH 6.6

DK

iayou...@gmail.com

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Aug 23, 2019, 6:15:05 AM8/23/19
to
I have a question.. Why is guanidine thiocyanate used for agarose gel solubilization (during the DNA gel extraction purification procedure) as opposed to guanidine hydrochloride?
after solubilization of the agarose gel, couldn't ethanol be used to precipitate the DNA? If my DNA sample is already very pure, I don't see what advantage there is to capturing the sample with silica gel resins?

Dr Engelbert Buxbaum

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Aug 23, 2019, 11:42:35 AM8/23/19
to
In article <e8985d36-8fd4-4dcf...@googlegroups.com>,
iayou...@gmail.com says...

> I have a question.. Why is guanidine thiocyanate used for agarose gel
> solubilization (during the DNA gel extraction purification procedure)
> as opposed to guanidine hydrochloride?

The thiocyanate ion SCN- is considerably more caotropic than chloride,
and hence more effective in solubilising agarose. The keyword to look up
is "Hofmeister-series".

--
DIN EN ISO 9241-13: 9.5.3 Error messages should convey what is wrong,
what corrective actions can be taken, and the cause of the error.

Bjorn

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Aug 25, 2019, 5:34:54 AM8/25/19
to
NaCl works well as a substitute. I have tried it myself.

https://www.oreilly.com/biocoder/issues/BioCoderSummer2014.pdf

https://github.com/cathalgarvey/biohacking-protocols/blob/master/Miniprep%20Using%20Diatomaceous%20Earth.md







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