Sincere thanks to everyone who responded to my query. What a great resource we have in evoldir! I have compiled the responses to date below. Each paragraph is a unique response. The consensus in the community is that the vast majority of people who are using the SABR SAFE system have been very pleased with the results. Many recommend incorporation directly into the gel. All noted that it is more expensive than using ethidium bromide, plus a new camera filter needs to be purchased, but some savings are re-captured by the fact that the gels do not have to be disposed of as hazardous waste (but some labs do so anyway, just to be sure). Most noted the light sensitivity of this stain. One lab manager directed me to her blog which raises the issue of whether ethidium bromide is really all that hazardous (http://rrresearch.blogspot.com/2006/10/heresy-about-ethidium-bromide.html). It does not work well or at all with RNA. Finally, a couple of labs are using GEL RED, another alternative, with good results. Alan ------------- Responses ------------------- Yes, I made the transition in my old lab. I found it great in a number of ways - in particular I found it to be more sensitive - I could see bands that were not observable using EtBr. Indeed, bands that you cannot see by eye also became apparent once viewed through the special camera-lense filter required. Which brings me to the bad points - you need to first buy a new lense filter as recommended, which is not cheap. And overall its more expensive than EtBr. But if you have the money I would recommend it - I never regretted the switch. We switched our laboratory over to Invitrogen's sybrsafe a year or two back. There are a couple of things probably worth noting. Firstly, it's a heap more light sensitive than EtBr so you can't pre-add the dye to agarose and store on the shelf for a while. This is not a big deal, it just means adding it fresh each time (or storing post stain solution away from light). I think the biggest thing is that it optimally fluoresces at a different wavelength from EtBr. We use a BioRad geldoc for visualisation and were originally using the same camera filter as for EtBr. This meant that the bands, although "see-able" were much fainter than with EtBr (and reasonably often, when bands on a gel were weak, we put it in EtBr for a while to get a decent view). Since then, we have purchased a different filter for Sybr and it's now a heap better, and I'd say every bit as good as EtBr. I'm totally happy with it, and even though it's a bit pricier than EtBr, I think it's worth it, and would totally recommend it (as long as you get a sybr filter for your camera). As an aside, Invitrogen market Sybr Safe as a totally "safe" alternative but I can't help but wonder if something that binds DNA can really be totally safe... i have been using the SYBR Safe since i moved to UBC six months ago. We use it to test PCR amplifications generally with 1.5 - 2 % agarose gel. The results are totally satisfactory with the recommended dosage (e.g. 9ul for 90ml gel). I guess it depends on what application you want to make of it, i heard some people still used ETBR as they found it more sensitive for other applications. we have been routinely using SYBR Safe for around 2 yrs now, and recommend it entirely... if you have 'very little' product though it may not be satisfactory, but in routine lab gels it should be an entirely useful replacement for ethidium bromide. Yes. My experience with SYBR Safe is entirely positive. It is more sensitive to timing your lab work than EtBr, i.e. the gel with SYBR Safe cannot be prepared long in advance and subsequently should be covered to prevent light exposition, but I was satisfied with it. We've been using SYBR Safe for a while now. We also bought Invitrogen's blue transilluminator to avoid using UV. Except of the price I think it is a good EtBr substitute. One thing though, it doesn't work with RNA, so we also kept the EtBr/UV facility. We have indeed tried out this stain as an alternative to Ethidium bromide and it works reasonably well. One drawback is that it is sensitive to light and once the stain is made up it does not last that long (<1 week). We have found a much better alternative called Gel Red nucleic acid stain which we buy from Jomar Diagnostics (your distributor may be different). This stain can be added to the gel prior to pouring and will survive repeated microwaving and cooling cycles. It is also not light sensitive, can be stored at room temp and seems to last for many weeks without any loss of signal. I would recommend it over SYBR safe. Hope this info helps. I'm currently working in a large genetics lab and we use SYBR DNA gel stain for all gels. Each aliquot of SYBR and TBE buffer can be re-used for up to 4 gels, and, on average, my gels look as good as they would with ethidium bromide. I have used SYBR GOLD recently, and it was fine. All it means is that you don't have to add anything to the gel and you add it to the samples when loading. Which helps if you want to re-use gels at any point. We visualised it using UV with no problems. Also, the SYBR GOLD itself needs to be kept away from light. The only drawback seemed to be when we saw little product and then we used EtBr to double check the samples. Yes, our lab is using SYBR Safe to stain Agarose gels and it works. But you can find several non important problems: the staining intensity decreases with time when you run the gel (run less than 60 min), when you store for a long time (months), and when you expose the gel for a long time to UV light. And of course it is much more expensive that Ethidium Bromide. But it works very well. We use 5 µl in 100 ml TBE Agarose gel. We have used GelRed with good success. Using post staining conditions we have reused the same staining bath up to 10x with good results. After this things start to deteriorate a bit. This compensates a little for the high cost - although of course it means everything takes much longer. has been great a great alternative... no problems! We are routinely using the invitrogen SYBR safe DNA stain since two months now. It works pretty well providing that you incorporate the SYBR safe directly into the agarose gel. Staining agarose gel in buffer bath with SYBR safe is not satisfactory compared to EBr. Also, you cannot reuse already stained and run agarose gels unlike with EBr. In routine, we add 5 ul of SYBR safe to 50ml agarose gel (1% with TAE 0.5x) in order to have fluorescent signals that are comparable to those obtained with EBr stained gels I hope that this will be helpful to you. Works great - we've used it for a year now. The dye is a bit more costly, but it is so much SAFER for both users and DNA if used with a blue light box. The blue transilluminator sold by Invitrogen has a design flaw that can be remedied by taping a sheet of acetate on top to prevent buffer from seeping in. I instigated and oversaw the switch from EtBr to SYBR-Safe during my time at the University of Edinburgh (Scotland). It is a perfectly good substitute, and in fact is often more sensitive than EtBr at DNA staining (as well as being safer). I ran comparative tests using single copy gene PCRs from various organisms (Drosophila, C. elegans, Silene, Arabidopsis) and found it performed well with everything I tested. Unfortunately I don't have the gel images anymore as I am now in Spain. Some people have pointed out a problem with the previous SYBR-Green, in that it migrated differently depending on the amount of product loaded onto a gel. However, this problem has been sorted out with SYBR-Safe. The only problem anyone could find with it was that it didn't work when using acrylamide gels - but given their safety record, and high quality agarose will do the same job anyway. My lab made the switch this past year and have not had any issues other than some problems when visualizing some RNA purifications that contain alot of pigments. It seems that the pigmenst may "quench" the Sybr safe dye. In those instances we use EtBR. In general I have noticed lower background when using sybr safe, which makes for nicer pictures. We use Sybr Green in our lab for certain applications. I'm not sure what the difference is between it and Sybr Safe, but I would imagine they are similar. We looked into using it as a substitute for EtBr because of the lower toxicity, but it was way too expensive. Also, because it is so much more sensitive, visualising our normal amount of PCR product was impossible (we typically use 1-5ng template DNA in a standard mitochondrial or microsatellite reaction)--it is a large smeary blob. For our low mass ladder, we have to dilute it 10 fold for it to be properly visible. Obviously one could dilute their product, or perhaps use less stain, but we opted to stick with EtBr for most applications. Another thing we found is that different amounts of product migrate at different rates. I found it somewhat accidentally as I was running a dilution series to test the sensitivity of sybr green...I think the result was that small amounts of product ran more slowly (it was awhile ago so I don't remember the exact details). There was a specific cutoff point of amount of PCR product where it shifted drastically...I don't remember what that limit was, but you could just test for it or keep in mind that the fragment size may be inaccurate when running low amounts of product. We usually already know the fragment size and are more concerned with product amount for sequencing and other downstream applications, so we can get away with it. All that said, we do use Sybr for low template reactions (generally <100pg template), and also for whole genomic gels since you can add much less stock DNA and still get a good visualisation. Another advantage to sybr is the stain doesn't migrate in the gel the way EtBr does (we add the stains directly rather than post-stain the gels). You might want to take a look at my blog post about this: http://rrresearch.blogspot.com/2006/10/heresy-about-ethidium-bromide.html We haven't used Invitrogen's Sybr Safe -- I'm guessing it's expensive -- but we occasionally use SYBR-Green in our gels (we also still use ethidium bromide), and have no problem with it, following the instructions that come with it. You should know that although it excites at about the same UV range as EthBr, its emission is different, so you need a different filter for your gel imager or camera. Our principal use of SYBR-Green is in real-time PCR. We buy it directly from Cambrex Biosciences, as we do another of their gel-staining products, FMC GelStar, which uses the same camera filter as SYBR-Green. I don't know what the safety issues are with this product, and we don't use it regularly or routinely, but only when its sensitivity and high signal/noise ratio are called for, when bands are far too faint for EthBr or SYBR-Green. We switched 2 or 3 years ago and have been happy with the stain. We do not use EthBr at all any more. This is Invitrogen's version of SYBR green. I initially switched over to using this stain (even though it is more expensive) due to its reduction of the hazmat disposal issue, but have been really pleased with the greater sensitivity of this stain over EtBr. I found this on the web: http://web.mit.edu/environment/pdf/SYBR.pdf Works fine after a bit of practice. Seems that you have to add the stain when the gel has cooled down, right before you are about to pour. We use it slightly diluted (6 syber: 2 1xTAE) and we put in 1.5ul for 15ml of gel. Yes, our lab is using SYBR Safe to stain Agarose gels and it works. But you can find several non important problems: the staining intensity decreases with time when you run the gel (run less than 60 min), when you store for a long time (months), and when you expose the gel for a long time to UV light. And of course it is much more expensive that Ethidium Bromide. But it works very well. We use 5 µl in 100 ml TBE Agarose gel. Our lab switched to it last year, and we've been very happy with it. In the past, we've reused gels (if someone pours a gel and only uses a few lanes, someone else uses the remainder of the gel later), but unfortunately we can't do that with SYBR Safe. The signal is also a little lower, which makes it more difficult to cut bands out of gels for gel extractions, but I haven't noticed any differences in the quality and intensity of signal in gel photos. I'm a little skeptical that it's truly safe, but my institution seems satisified. Not having to deal with hazardous waste is a big plus! We switched to SYBRsafe a couple of years ago... Gel bands are just as clear, or slightly clearer, than with EtBr. Obviously we have no data on how much less mutagenic SYBRsafe is, but it's not very expensive and works fine. Yes, we use it as we had EtBr and it's very effective. Our environmental health & safety people suggest treating it like EtBr waste -- although Invitrogen's claim is that it's safer. We add it directly to cooling gels, rather than doing a bath for staining. Syber Safe works well for us visualizing PCR products, low yield extractions, and genomic amplifications, AFLP pre-amps, ... My old lab used SYBR green for staining SSCP gels (a kind of acrylamide). It worked well enough for that, but we knew we were loading plenty of DNA having run the reactions out on agarose and stained with EtBr to test them. The gels were also only a couple of milimetres thick. We have used SYBR safe in place of EtBr for quite some time now. It works very well and many appreciate the non-carcinogenic aspect!! We use Sybr Safe in Chris Simon's lab at UConn. We are generally happy with the results, though it does not seem as durable as ETBR. We cast it into our gels, though I am experimenting with including it in my loading dye. My comment about "durability" comes from my impression is that it fades after exposure to strong UV, so we keep the Sybr stocks in the dark (our lights are fluorescent), and we cast the gels in the dark. I also let the molten agarose cool off just a bit before adding the stain. Other than that, there was no particular difficulty in switching from ETBR to Sybr Safe. On the plus side, it doesn't seem to cause any "background glow" when cast into a gel, and it doesn't seem to run out of the gel and into the anode buffer in the way that ETBR does. We dispose of our used gels in the hazardous waste. Although the MSDS for Sybr safe doesn't say that the stuff is particularly hazardous, it doesn't say that it's safe, either. My thoughts are that it *does* bind to DNA, and that makes it a potential mutagen, so we treat Sybr Safe as if it were just as hazardous at ETBR. Even so, we feel a little more comfortable working with it than we did with ETBR. -------- PLEASE NOTE THE NEW PHONE NUMBER! Alan W. Meerow, Ph.D., Research Geneticist and Systematist USDA-ARS-SHRS, National Germplasm Repository 13601 Old Cutler Road, Miami, FL 33158 USA voice: 786-573-7075; FAX: 786-573-7110 email: alan.meerow@ars.usda.gov "Meerow, Alan"