Dear EvoDir members: Please, find here below a summary of the answers I got from all of you about question on DNA stain. Thank you very much for your very useful suggestions! Cheers, Filippo ----- 1. The idea that Ethidium bromide is dangerous, especially at the quantities used to stain gels, is probably false, and is certainly not based on sound mechanism nor strong epidemiological evidence. The article below is very interesting: http://goo.gl/AJKSup For staining DNA on agarose, our labs use Gel red which is no where as toxic as ethidium bromide http://www.bioscience.co.uk/site/user/ page.phtml?page_id 22&search=gel+red&productgrp=OTHER&sid=tgtk74337 Did you try SYBR Safe from Invitrogen? We have been using it for the past 9 months now and it is really reliable (we have even reduced the concentration by half). 4. We use a product called Gel Red. It works well. Not sure who would carry it in Europe, but VWR carries it in the states: http://biotium.com/technology/gelred-gelgreen-nucleic-acid-gel-stains/ 5. Maybe GelRed could help you (http://biotium.com/technology/ gelred-gelgreen-nucleic-acid-gel-stains/). During my last internship, I used it. It is supposed to be non-toxic, but my supervisor was not sure that degradation pruducts were non-toxic. Have you tried RedSafe? Here is as example: http://www.chembio.co.uk/ product_detail.php?product_id0 We use Midori green it's just as good if not better than ethidium bromide and less toxic. You may find this blog post helpful: http:// rrresearch.fieldofscience.com/2006/10/heresy-about-ethidium-bromide.html 9. We use GelRed from Biotium. It is quite bright and works with the same filters as ethidium bromide. 10. We use EZ-Vision: http://www.amresco-inc.com/home/products/best- sellers/EZ-Vision.cmsx I have been using a product called SafeView by NBS biologicals: http://www.nbsbio.co.uk/nbs-sv1 12. In our population genetics lab, we use a product called GelRed, manufactured by Biotium. It gives us very good results. Bands are easily visible on the 1% and 2% agarose gels we use. We use the standard safety and handling procedures as those with ethidium bromide (gloves, gelred only glassware, contamination area, etc), but it is reported to be non-toxic and non-mutagenic. I hope this helps! I use EZ-vision by Amresco. The RNA version also works. We use SafeView (www.nbsbio.co.uk/nbs-sv1) and have never had any problems! My lab uses SYBR Safe with a Blue-Light source. It works nearly as well as Ethidium and is much safer: http://www.lifetechnologies.com/ us/en/home/life-science/dna-rna-purification-analysis/nucleic-acid- gel-electrophoresis/dna-stains/sybr-safe.html 16. We use SybrSafe - it works nearly as well as EtBr: http:// www.lifetechnologies.com/us/en/home/life-science/dna-rna-purification- analysis/nucleic-acid-gel-electrophoresis/dna-stains/sybr-safe.html Try Nancy-520 from Sigma: http://www.sigmaaldrich.com/life-science/ cell-biology/detection/learning-center/nancy-520.html Nancy seems like a nice alternative to SYBR and ethidium bromide, but I've never used it. We've had success with SYBR Safe DNA gel stain for >7 years in the Johnson Lab, and, given it produces comparable results while being much safer than ethidium bromide, several other labs use it here on campus at BYU. If there is a chance you could stick with SYBR, maybe you could explain the problem in further detail, and then people could help you arrive at a solution with SYBR. So far, it remains unclear why these other options haven't worked for you. We use a product called GelRed from Biotum. It works quite wells as an "in-gel" or "post-gel" stain. http://biotium.com/technology/gelred- gelgreen-nucleic-acid-gel-stains/ We use the GelRed Nucleic Acid Gel Stain from Biotium (http:// biotium.com/product/gelredtm-nucleic-acid-gel-stain-10000x-in-water/) and we have satisfactory results. Here is our protocol: Mix 1 4 mg of BFB (Bromophenol blue) 1 ml of mili-Q Water Mix 2 174 ul of Mix 1 250 ul of glycerol 576 ul of mili-Q Water Loading Solution 1 ul of GEL RED 999 ul of Mix 2 Use 1 ul of Loading Solution for stain 3 ul of DNA. GelRed works well, though despite claims of being non-cytotoxic etc., I am wary of any substance that binds DNA and use gloves etc. for protection. No new camera filter is required to visualize DNA. We use peqGreen, that works quite well for us. Occasionally, one has to use a little bigger amount than stated in the manual, but then it works fine. Advantage is that you can use the same filter as for EtBr, so no need to adapt your image system if you worked with EtBr so far! See attachments for details. It might depend on what you want to use the dye for if it works for you or not, I guess ˇV we use it mostly for controlling if PCRs have worked etc, so no quantification, reallyˇK. We add the dye directly to the agarose gel, after it has cooled down a bit after boiling, before pouring and loading it. 24. I was using Nancy-520 to stain DNA on gel. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/ Datasheet/6/01494dat.pdf http://www.sigmaaldrich.com/life-science/cell-biology/detection/ learning-center/nancy-520.html We added 10 ul of Nancy-520 to our agarose (TAE or TBE) 100 mL warm solution. Always we had great results. At the moment I am testing new products and I am very interested in this subject also. I've decided to try : Midori Green ( Nippon Genetics) - added to gel Novel Green ( GeneDirex) - added to gel USB (SynGene) - added to sample The best results were for USB but I don't want to add anything to samples, especially when I have a big PCR reaction, so now I am testing these two others We use GelRed from Biotium in out lab, with good success. We order it through Fisher Scientific, so it should be easy for you to find, if you want to check it out. MY NEW EMAIL ADDRESS IS: filippo.barbanera@unipi.it ***** Filippo Barbanera Researcher Department of Biology Zoology and Anthropology Unit Via A. Volta, 6 I - 56126 Pisa (PI) Italy Web site: www.biologia.unipi.it room: + 39 050 2211386 lab. : + 39 050 2211343 fax: + 39 050 2211393 ***** FILIPPO BARBANERA