14 replies to this topic

[desperado]

Most people use Ethidium Bromide (EtBr) as a fluorescent dye for DNA. People know it is poisonous and dangerous to cause cancer. Also people use UV to excite EtBr, which is risky for eyes and skins. Some people start to use safe dyes, like SyBr Safe, Gelgreen, Gelred, etc. They are safer but more expensive than EtBr, and can be excited with visible light which is much safer. There is MaestroGen UltraBright LED transilluminator that can be used as a benchtop instrument for these safe dyes. However, our lab is still using Etbr. Just wanna know what other people’s opinion about using safe dyes instead. Cost oriented or safety oriented?

[hobglobin]

The real toxicity and hazard of EtBr seems still questionable, though all the classifications (MSDS et al) classify it this way. And I also agree that one should be careful and wear gloves, and don't use powders of it. Anyway there's also some hysteria about this substance, read this article about it. An useful and reasonable text.
Anyway many of the dyes are more or less dangerous as they all interact in some way with your DNA and can penetrate through your skin and gloves in different speeds, or are later metabolised to toxic products. Here is an unmasking blog about SYBRsafe...
I guess silver is a quite safe alternative (though the developer and fixation substances might be not). Crystal violet and some other dyes might be also an alternative with less toxicity, read this pdf.
Read also this thread from here. Swanny convinced me somehow...

Edited by hobglobin, 22 October 2010 - 11:03 AM.

[desperado]

I found a video of MaestroGen UltraBright LED transilluminator for DNA gel cutting. This instrument looks not bad!

[donny]

EtBr is cheaper but the "safer" dyes can be more sensitive. You may save by using less DNA ladder. Also, the link for the LED illuminator says it works for EtBr too. Do you know the price of the LED illuminator relative to UV ones? We are getting one for the lab and I might suggest the LED one instead if the price is right.

[perneseblue]

desperado, on 22 October 2010 - 08:19 AM, said:

People know it is poisonous and dangerous to cause cancer

Actually that is not true.
To the best of our knowledge EtBr has not been shown to cause cancer in cow which have ingested EtBr. Nor have increase levels of  birth defect been detected in calf born from cow which have ingested EtBr.

It should also be known that EtBr aka Hofnium was once use as a drug to treat sleeping disease (Trypanosoma brucei). And it is still used in that capacity to treat life stock in africa.

EtBr has been noted to cause mutation in E. coli and that is where all the worry comes from. But on the same note caffeine is known to cause chromosomal lesions and aberrations in sister-chromatid exchange in eukaryotic cells (including human cells). Yet we have Starbucks. So we have to put the risk in perspective.

Unfortunately we do not have the data to show how dangerous EtBr is to humans. Similarly I have doubts about the actually safety of dyes such as SyBr Safe, Gelgreen, Gelred, etc. They too intercalate into DNA and we have no real data to prove how safe these compounds are.

Thus we cannot even begin to debate cost verses safety as we don't know how dangerous EtBR is compared to SyBr Safe, Gelgreen etc. For all we know some of the more expensive DNA stains maybe more dangerous than EtBr. At least EtBr has a history being used as a drug.

[UBClabbie]

Safe dyes are added only to the sample and not to the gel. This means that you won't have any streaks or unwanted feedback when you go to visualize your gel.
It also means you don't have the extra risk of contaminating glassware and other equipment in your lab. So, the risk is more "contained"

As well, I'm not sure if safe dyes are technically safer for the environment, since they are non-carcinogenic opposed to EtBr which is generally thought of as a carcinogen.

Some safe dye examples
https://www.abmgood....r-SafeView1.pdf

desperado, on 22 October 2010 - 08:19 AM, said:

Most people use Ethidium Bromide (EtBr) as a fluorescent dye for DNA. People know it is poisonous and dangerous to cause cancer. Also people use UV to excite EtBr, which is risky for eyes and skins. Some people start to use safe dyes, like SyBr Safe, Gelgreen, Gelred, etc. They are safer but more expensive than EtBr, and can be excited with visible light which is much safer. There is MaestroGen UltraBright LED transilluminator that can be used as a benchtop instrument for these safe dyes. However, our lab is still using Etbr. Just wanna know what other people’s opinion about using safe dyes instead. Cost oriented or safety oriented?

[mdfenko]

biotechgirl, on 08 November 2010 - 12:49 PM, said:

Safe dyes are added only to the sample and not to the gel. This means that you won't have any streaks or unwanted feedback when you go to visualize your gel.
It also means you don't have the extra risk of contaminating glassware and other equipment in your lab. So, the risk is more "contained"

As well, I'm not sure if safe dyes are technically safer for the environment, since they are non-carcinogenic opposed to EtBr which is generally thought of as a carcinogen.

have you read through this thread?

and

if the sample streaks then the stained sample will streak whether the stain is added to the sample, gel or buffer.

[HomeBrew]

perneseblue, on 07 November 2010 - 09:31 AM, said:

Unfortunately we do not have the data to show how dangerous EtBr is to humans.

All the empirical data we have says it's not dangerous.  This is a chemical used to stain gels daily in tens of thousands of labs around the world for decades by everyone from seasoned PIs to summer students -- have you seen a single report of a death or illness being caused by it?

[UBClabbie]

mdfenko, on 09 November 2010 - 11:39 AM, said:

biotechgirl, on 08 November 2010 - 12:49 PM, said:

Safe dyes are added only to the sample and not to the gel. This means that you won't have any streaks or unwanted feedback when you go to visualize your gel.
It also means you don't have the extra risk of contaminating glassware and other equipment in your lab. So, the risk is more "contained"

As well, I'm not sure if safe dyes are technically safer for the environment, since they are non-carcinogenic opposed to EtBr which is generally thought of as a carcinogen.

have you read through this thread?

and

if the sample streaks then the stained sample will streak whether the stain is added to the sample, gel or buffer.

i'm not talking about the sample streaking, i'm talking about streaks because the EtBr is improperly distrubuted throughout the gel. Sometimes cross contamination on your gels or other equipment causes "flashes" of light on your gel when you go to view it. Streaking on your gel fromthe sample is due to degradation.. I'm talking about something entirely different....

And yes I read this thread... I'm pointing out that even if EtBr isn't a carcinogen, it might still have other harmful effects and essentially your EtBr still touches more glassware than safe dyes....

[seanspotatobusiness]

Why can't you add EtBr directly to samples before loading, supposing it's convenient (e.g. a loading dye which also contains EtBr)?

[Adrian K]

seanspotatobusiness, on 10 November 2010 - 07:55 AM, said:

Why can't you add EtBr directly to samples before loading, supposing it's convenient (e.g. a loading dye which also contains EtBr)?

Yeah, good question. If you run EtBr pre-stained gel before, you will know the EtBr will move towards the opposite direction with your nucleic acid. So, if you mix it to your sample with loading dye, you will see nothing in the end because EtBr had moved out of the gel.

Unless somebody proves me wrong...

[hobglobin]

adrian kohsf, on 10 November 2010 - 08:41 AM, said:

seanspotatobusiness, on 10 November 2010 - 07:55 AM, said:

Why can't you add EtBr directly to samples before loading, supposing it's convenient (e.g. a loading dye which also contains EtBr)?

Yeah, good question. If you run EtBr pre-stained gel before, you will know the EtBr will move towards the opposite direction with your nucleic acid. So, if you mix it to your sample with loading dye, you will see nothing in the end because EtBr had moved out of the gel.

Unless somebody proves me wrong...

Well it is suggested sometimes (here) and you even can buy ready-made loading buffers containing Etbr, e.g. here, though I guess this is a waste of money...
This paper may help, though it is not online:
Angermüller SA, Sayavedra-Soto LA: Rapid visualization of genomic DNA and total RNA in agarose gels. Biotechniques. 1990 Jan;8(1): 36-38.
If someone has it as pdf, I'd like to have a copy...

[HomeBrew]

adrian kohsf, on 10 November 2010 - 08:41 AM, said:

If you run EtBr pre-stained gel before, you will know the EtBr will move towards the opposite direction with your nucleic acid.

This is undoubtedly true for EtBr that is not associated with DNA, but I'm pretty sure once it's intercalated, it'd be carried along with the DNA molecule...

[bob1]

The intercalated EtBr is carried along with the DNA, but it does retard migration a bit so it is not good to do if you want to compare sizes of migration accurately.

[Adrian K]

Wow, interesting. If this is the case, I will try to run a gel tomorrow, one without EtBr stained but add EtBr in the sample buffer; another as normal EtBr stained gel...
will let you guys know the result.