Biomakespace Forum

Ethidium Bromide Alternatives


(om259) #1

Hello,

I thought I’ll put the information I have here as we looked into it recently and now my lab uses Gel Red and SYBR Gold. As you can see the prices are quite high compared to EtBr but at least they are similar across the alternative products:

EtBr (Sigma)
£45 for 10ml (£4.50 per ml)

Nancy-250 (Sigma)
£99 for 0.5ml (£200 per ml)

GelRed (Cambridge Bioscience)
£98 for 0.5ml (£200 per ml)
or
£1852 for 10ml (£185 per ml)
(in water, slightly more expensive in DMSO)

Sybr Safe (ThermoFisher)
£53 for 0.4ml (£133 per ml)

Sybr Gold (ThermoFisher)
£118 for 0.5ml (£236 per ml)

We have not tested Nancy-250. Gel Red works perfectly well for most gel based PCRs. We only use Sybr Gold sometimes do distinguish small bands.
SybrSafe seems to be the cheapest alternative.


(Matt) #2

Really useful to know, thanks for this! Quite pricy though!

I use Hoechst stain all the time for staining the genomic DNA in nuclei. Its super cheap, I get the powder for £15 from Sigma (33342 variant), I’m still on my first bottle since before Dec 2015!

There is a paper here describing using Hoechst 33258 for DNA gels, and I have to say I’m pretty impressed with the gel pictures, not to mention the fact they’re from 1986!


(om259) #3

As far as I know there is some debate on how safe (non-mutagenic or toxic) Hoechst is. We need to look into it further. It does have pretty harsh annotation by Sigma (H302-H315-H319) but it might be an incremental improvement of EtBr :slight_smile:


(Jenny Molloy) #4

Interesting article on 'The Myth of EtBR’

I have also been told by various people that the evidence doesn’t back up the feeling that it is one of the more dangerous things in a molecular biology lab, but that doesn’t necessarily mean we shouldn’t go for SYBR Safe anyway. It’ll be interesting to hear the University Safety Office perspective! We do of course have more issues with disposal than most labs, so that may be reason enough in itself.


(Emma J) #5

Hi All,

I’ve read that article before - though I don’t really see any need for
using EthBr where there are safer alternatives. The lab I work in still
uses quite a bit of EthBr, but I’ve taken to using Sybrsafe for gel
stains (post) - it has worked very well for me so far. As far as I’m
aware Hoechst too is fairly toxic.

Emma


(Matt) #6

Useful to know different points of view. The sigma documentation is probably most severe for the powder, hopefully less for the liquid, but I’m not sure.

Blue light for visualisation is a big big plus for sybr safe better for people DNA and plasmid DNA!

I’ll place myself in the SybrSafe camp now :slight_smile:


(Matt) #7

I was just looking into how to dispose of Hoechst and I found this forum post:
http://www.bio.net/mm/methods/1997-February/054778.html

it references some papers that apparently found that Hoechst binds to the outside of the double helix and does not intercalate between DNA bases, which supposedly makes it much safer than EtBr.

Looking at Sigma Hazard info it has the following flags:
H302 - harmful if swallowed
H315 - causes skin irritation
H335 - may cause respiratory irritation
H341 - suspected of causing genetic defects (germ cell mutagenicity)

I think using at low concentrations and standard PPE hopefully protect against all of these risks (at least I hope so because I’m using it a lot!).

We will have to have some DNA dyes in the lab if we’re going to have people doing microscopy on cells, so it could still be worth considering Hoechst as a cheap option for staining gels (especially if we’ll have to do safety documentation for it anyway).


(Dean Madden) #8

I think there are three sorts of stain for DNA: those that intercalate within the double helix (like ethidium bromide), those that bind to the outside of the helix (e.g., methylene blue, Azure A, Toluidine blue O and Nile blue sulphate) and molecules that bind to the DNA but are too big to pass through cell membranes (e.g., Megafluor). The intercalators are thought to cause frameshift mutations, and are considered (rightly or wrongly) the most harmful while the others are generally thought to be safer.

There is some dispute about whether some thiazin dyes like Methylene blue actually intercalate (I’ve seen a not very convincing paper suggesting that this might happen). There have been a couple of useful surveys of various stains e.g., Adkins, S. and Burmeister, M. (1996) Visualization of DNA in agarose gels as migrating colored bands: Applications for preparative gels and
educational demonstrations. Analytical Biochemistry 240, 17–23.

Bear in mind when looking at Safety Data Sheets that they may refer to the powder or whatever is supplied rather than the dilute solution you end up using on the gel. So for example, Toluidine blue O as a powder is potentially harmful, whereas the dilute (0.04% w/v) aqueous solution you might use is not thought to be harmful.


(Dean Madden) #9

Here are some other relevant papers for cheap gel stains. Be warned that some of these stains are not particularly good. For instance, methylene blue usually requires de-staining to remove the background colour from the gel, plus it needs a lot more DNA in the gel – and you won’t see the bands of small fragments easily. The best stains of this type I’ve used are Toluidine blue O and Nile blue sulphate, btw.

Brilliant cresyl blue
Santillán Torres, J. and Ponce- Noyoia,P. (1993) A novel stain for DNA in agarose gels Trends in Genetics 9 (2) 40.
We once tried this with a 40-year-old pot of Brilliant cresyl blue and it worked exactly as described in this paper. New stuff, however, didn’t work at all. We tried all sorts of tricks to artificially age the new dye but with no success.

Methylene blue
Yung-Sharp, D. and Kumar, R. (1989) Protocols for the visualisation of DNA in electrophoretic gels by a safe and inexpensive alternative to ethidium bromide. Technique 1 (3) 183–187.


(Dean Madden) #10

You can also use some of these thiazin dyes to enable the DNA to be seen as it moves across the gel. Nile blue sulphate (also known as Nile blue A) can be used in this way. Adkins and Burmeister (mentioned above) give useful guidance as to its use as well as hints for identifying other dyes which may be useful for visualising DNA.

All of these dyes used for staining ‘mobile’ DNA are cationic — that is, they are positively charged in the gel buffer, at pH 8. They move through the gel in the opposite direction to the DNA, latching onto the DNA molecules as they meet them. So that sufficient dye remains in the gel, it is added to both the gel and the buffer above it. However, a far lower concentration (1–3 μg per mL) of dye is necessary for this method than for post-electrophoresis staining. This is because too much dye will neutralise the negatively-charged DNA fragments, slowing their movement and reducing the resolution or even preventing the DNA from moving at all. Consequently, there is a compromise to be struck between visibility and resolution. Better results are usually achieved by staining the DNA after the gel has been run, rather than staining during the run.


(Matt) #11

Thanks for the really helpful insights and links Dean!


(Miha Pipan) #12

Hi there,

I know EtBr is a touchy subject, but let’s face it: one should pay extra care when using any of these potentially harmful DNA dyes, not just EtBr. The only added step of EtBr work is the appropriate disposal of contaminated gels/waste, which can be a pain in a small lab like mine, but should not be quite as much of a problem in a large-volume community lab.

From a purely financial perspective, EtBr is the cheapest sensitive stain one can get atm. I bought 1g for £30 a few months ago, and will probably own this for probs the next decade given the gel gets a ~1:10.000/20.000 dilution of a 10mg/mL stock. All staining should be performed with PPE anyway, and as long as these and the gels, tips etc. are disposed of in a dedicated bin, there should be little issue, especially if an activated charcoal step is added before disposal.

I’d be also happy to donate 10 mL of such solution, so the cost is really zero at this stage. As sponsorship/grants accumulate, maybe Thermo won’t have an issue forking out a litre of SybrSAFE down the line, but I have a hunch - and I may be wrong!; that there are other investments that may need priority over spending £200+ on gel dyes.

Miha


(Dean Madden) #13

The other safety issue with many fluorescent dyes (although not all of them) is the harmful UV light from a transilluminator. Even blue (non-UV, say about 470 nm) can be harmful to the retina if the intensity is great enough and you stare at it. Apparently this is of particular concern with children. http://www.electronicsweekly.com/news/products/led/ensuring-safety-in-led-lighting-2012-11/