Gel Red questions

[Jeswin]

I'm looking into using Gel Red dye instead of EtBr. They have
different versions. I think the 0.5 mL 10000x in water stock solution
(catalog # 41003) is what I should be getting.

I'm wondering how I would use it. It says use at 1:10000 dilution so
does that mean use 0.1uL per 1mL gel volume? If I have a 50mL gel, do
I add 5uL gel red? That would give me 100 gels. Hmm....for $100. Seems
expensive.

They also have 4L of 3x in water for $200. They say dilute to 1x but
don't say what concentration to use in a gel. What do you guys do?

Thanks

[Dakota Hamill]

I use 5 uL in a 50mL gel, though I use GelGreen, but same difference.  They both come at 10,000X though, so like you said, to get to working concentration do a 1:10k dilution.  It comes out to about 95 cents or 1 dollar per gel, and I got the 100uL size for $21.

Knowing I don't have to deal with EtBR waste disposal is awesome, and if you factored in the cost of that, it'd probably still be cheaper to use GelRed or GelGreen.

[Jeswin]

On Tue, Mar 12, 2013 at 11:07 AM, Dakota Hamill <dko...@gmail.com> wrote:
>
> Knowing I don't have to deal with EtBR waste disposal is awesome, and if you
> factored in the cost of that, it'd probably still be cheaper to use GelRed
> or GelGreen.
>

Yes, that is the main reason I'm looking at this.

Any idea how to use the 3x in water stock gel red? The protocol is here:
http://www.biotium.com/product/product_info/Protocol/PI-41001.pdf

[shamrock]

i use Gel Red at a 1:10,000 dilution which means 1 microliter for every 10 milliliters of gel volume. I put it into the gel itself instead of staining after ward. More expensive than EtBr-yes, but in my mind worth it. It also seems to be more sensitive than EtBr, so you could possibly get away with a higher dilution (something to look into). 

[Nathan McCorkle]

On Tue, Mar 12, 2013 at 8:42 AM, Jeswin <phill...@gmail.com> wrote:
> Any idea how to use the 3x in water stock gel red? The protocol is here:
> http://www.biotium.com/product/product_info/Protocol/PI-41001.pdf

It says how to use it in the protocol, on the right middle/lower side.

Either use 3X for post-staining gels, at least 2-3 gels worth of
post-stains per volume (assuming you need at least enough liquid to
cover or keep the gel immersed)
or
for 60mL gel Use 20mL 3X with 6mL 10X buffer and 34mL dH20


--
-Nathan

[Jeswin]

Hey Nate, I saw that for preparation but how much per gel? 5uL? Is the
1x final solution at 1:10000 dilution? I don't do post staining; only
precasting.

[Nathan McCorkle]

On Tue, Mar 12, 2013 at 10:10 AM, Jeswin <phill...@gmail.com> wrote:
> Hey Nate, I saw that for preparation but how much per gel? 5uL? Is the
> 1x final solution at 1:10000 dilution? I don't do post staining; only
> precasting.

If you don't do post-staining then you always want the working
concentration to finally be 1X. If the solution starts as *3X*
concentration:
for 60mL gel add: 20mL *3X*, 6mL *10X* buffer and 34mL dH20

If the starting solution is *10000X*
for a 50mL gel:
50mL * 1000uL/mL / 10000X = 5uL *10000X*
49.995 mL 1X buffer

--
-Nathan

[Nathan McCorkle]

Yeah, that's why big companies and universities don't switch.

EtBr is molecularly known (no one really knows exactly what the gelRed
and sybrSafe molecules are, we know what classes they're in or one
that was described in one patent, etc)
so it's more 'open' in that sense, that would also probably make it
more reliably modeled in-silico (it's a much simpler molecule as well)

On Tue, Mar 12, 2013 at 11:57 AM, Jeswin <phill...@gmail.com> wrote:
> Hmm, that's what I thought. It seemed like too much. For my 1% EtBr
> gels, its 50mL 1x TAE + 0.5g agarose + 1uL EtBr.
>
> I got enough EtBr to last 100 years at the rate I use.



--
-Nathan

[Avery louie]

Also EtBr requires UV to view- gelgreen can be excited with a few blue LEDs, making it cheaper up front if you want to run a gel.

--A

--
-Nathan

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[Eugen Leitl]

On Tue, Mar 12, 2013 at 03:19:23PM -0400, Avery louie wrote:
> Also EtBr requires UV to view- gelgreen can be excited with a few blue
> LEDs, making it cheaper up front if you want to run a gel.

EtBr is overdemonized. Many reagents chemists used to work with
make it look like weak mouthwash.

Anything detecting DNA will likely intercalate, and do
pi-pi complex things in order to be visible, so it
almost certainly has an impact on base reading/frame shift,
and hence more or less mutagenic.

[Jeswin]

On Tue, Mar 12, 2013 at 4:04 PM, Eugen Leitl <eu...@leitl.org> wrote:
>
> EtBr is overdemonized. Many reagents chemists used to work with
> make it look like weak mouthwash.
>

If you can't put it in regular waste, then it makes no difference.
The gov't says "no way" so what are we little guys supposed to do?

I mean, it's a pretty strong DNA intercalator. I only use 1uL EtBr in
a 50mL gel and get really bright bands. The Gel Red dye says it needs
16mL for a 50mL gel. That's a lot of Gel Red compared with EtBr. I
assume you get the same results. Am I right?

[Nathan McCorkle]

On Tue, Mar 12, 2013 at 3:34 PM, Jeswin <phill...@gmail.com> wrote:
> On Tue, Mar 12, 2013 at 4:04 PM, Eugen Leitl <eu...@leitl.org> wrote:
>>
>> EtBr is overdemonized. Many reagents chemists used to work with
>> make it look like weak mouthwash.
>>
>
> If you can't put it in regular waste, then it makes no difference.
> The gov't says "no way" so what are we little guys supposed to do?
>

Could you just throw it in a warm oven to dry it, then wait til you
have a lot and find some company to take it from you?

> I mean, it's a pretty strong DNA intercalator. I only use 1uL EtBr in
> a 50mL gel and get really bright bands. The Gel Red dye says it needs
> 16mL for a 50mL gel. That's a lot of Gel Red compared with EtBr. I
> assume you get the same results. Am I right?

Not really, since the 3X is simply 10000X diluted in dH2O (that's why
they sell both, the former being more expensive but more convenient if
you're.... I'm not sure why you'd buy that one, anyway...)

Also the claimed sensitivity of gelGreen over EtBr is better (they
claim 0.1ug but don't give dimensions mm^2 or mm^3)

--
-Nathan

[Avery louie]

16ml?  do you mean ul?  I use 5ul gelgreen 10,000x for my 50ml gels, which is about a dollar a gel.

[Jeswin]

On Tue, Mar 12, 2013 at 8:11 PM, Avery louie <inact...@gmail.com> wrote:
> 16ml? do you mean ul? I use 5ul gelgreen 10,000x for my 50ml gels, which
> is about a dollar a gel.
>

They also sell 4L of 3x Dye for an extra $100. That kind of confused
me at first.

[Teshome Yehualaeshet]

What is the actual concentration of GelRed in mg/ml or other formulation ???

[Mega [Andreas Stuermer]]

Now that would be a project. Masspec of gelred and hacking it DIY

[John Ladasky]

On Tuesday, March 12, 2013 at 12:03:42 PM UTC-7, Nathan McCorkle wrote:

Yeah, that's why big companies and universities don't switch.

EtBr is molecularly known (no one really knows exactly what the gelRed
and sybrSafe molecules are, we know what classes they're in or one
that was described in one patent, etc)

I'm pretty sure that this information cannot be kept secret.  To obtain a chemistry patent, and to publish an accurate MSDS, the structure of the novel active compound must be known and disclosed.

I remember that SybrSafe is in the cyanine dye family, a descendant of Thiazole Orange.  If you look at the Wikipedia article for SybrSafe, a structure is published, and it sure looks like a cyanine dye to me.

https://en.wikipedia.org/wiki/SYBR_Safe

 

Wikipedia entries are also available for GelGreen and GelRed.  They look like acridine orange and ethidium dimers, respectively, with a peptide-ether linker between the individual fluorophores.

https://en.wikipedia.org/wiki/GelGreen

https://en.wikipedia.org/wiki/GelRed

The idea of making ethidium dimers is not new.  An ethidium dimer was available in 1990.  It's still available.  The linker differs from the GelRed linker, but otherwise the two molecules are quite similar.

https://www.thermofisher.com/order/catalog/product/E1169

[Mega [Andreas Stuermer]]

So are there cheaper sources?

http://www.sigmaaldrich.com/catalog/product/sial/390062?lang=de&region=AT 

Like this

[Dennis Oleksyuk]

It is much cheaper here if you buy 5G at a time.

http://www.adipogen.com/cdx-t0013/thiazol-orange.html

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[Mega [Andreas Stuermer]]

I dont see the prize... It says INQ-AT

[Dorif]

Well, it is quite easy to make these substancies known. Just purify them by thin-layer chromatography and make NMR analysis for every spot on the chromatograme. I have access both to the TLC and NMR spectrometer, but have no GelRed or SYBR Safe, but I think here are a lot of people, who have access to these dyes, TLC and NMR. Why not make GelRed and SYBR Safe more known?)

[John Ladasky]

SYBR Safe and Thiazole Orange are very similar, but not identical.  So is Thiazole Orange an acceptable substitute for the more expensive SYBR Safe in gel staining?  If so, why isn't everyone using it?

Thiazole Orange is membrane-permeable.  It was first developed to label RNA in intact reticulocytes.  I'm pretty sure that Thiazole Orange also binds DNA.  All DNA intercalating dyes are suspected carcinogens, for obvious reasons.

One way that SYBR Safe might be "safer" than Thiazole Orange is if the former was membrane-impermeant.  Is it?  I doubt it.  If anything, I would expect SYBR Safe to be slightly more membrane-permeant, because SYBR Safe has a propyl group where Thiazole Orange has a methyl group.  However, that structural change might also reduce toxicity.

I just had a look at an MSDS for each compound, and I didn't see any obvious differences.  

[Ra Ramana]

For those interested, I came across this dye, based on a food additive.

http://www.tintorang.com/

This alone caught my attention:

tinto rang™ claims to be 100% safest, as it is a plant based food grade product, with zero toxicity, mutagenicity and is non-hazardous.

It will solve the issues with EtBr. 

http://www.tintorang.com/Nucleic-Acid-Gel-Stain/tinto-rang-safe-nucleic-acid-gel-stain/safe-food-grade-nucleic-acid-DNA-RNA-Fluorescent-Dyes.html

-Ravi