Does anyone add Etbr to running buffer used to make agarose gels? - (Nov/13/2006 )
I have questions that I hope someone can answer. In our lab we currently stain/destain agarose gels with Etbr, but we would like to move to a system where we just add Etbr to our 15 liter container of TAE buffer. We use this container for running buffer and gel preparation. If anyone is in a lab currently using this practice my questions are: 1. how much Etbr/liter should I add? 2. Is Etbr effected by the process of boiling in the microwave during agarose gel preparation? Thanks for your time.
i don't think this will be a good idea to add EtBr into running buffer. EtBr will probably not be affected by boiling but you, your lab mates and the microwave will be. it is a carcinogen reagent so u must use it carefully.
that sounds like a bad idea.
add it to your agarose after nuking it in the microwave, after cooling the agarose down a little. it's not a time consuming step.
EtBr is also light sensitive, and most lab carboys are not light proof.
Stain/destain is indeed a pain in the butt - so do what the others suggested. Keep a stock solution of EtBr (10 mg/ml) and add a little (one microliter) to 100 mls of melted agarose, just before you pour the gel.
Stain/destain is indeed a pain in the butt - so do what the others suggested. Keep a stock solution of EtBr (10 mg/ml) and add a little (one microliter) to 100 mls of melted agarose, just before you pour the gel.
I am convinced. I had a feeling there was a reason why I should not do it the way I had proposed, I just needed to have my intuition corroborated. I would like to thank you all very much for the good advice.
I'm going to be the voice of disagreement on this. We routinely make up 1 lilter of TBE for gel prep/running buffer. We add 15uL of 10mg/mL Ethidium Bromide to the solution and leave it on the counter in a clear flask.
This solution is used to make the gels and for running buffer as needed. Since TBE is more reusable than TAE, we have a flask of used TBE for running buffer, but this is supplemented by the fresh stocks whenever more is needed. We've never had a problem with poor running conditions or experienced problems with microwaving the EtBr.
I guess I'm in the minority on this one, but I thought I'd let you know it is possible and it does work. 
I had microwaved the agarose gel containing EtBr...no problem...as long as I don't overheat it and spill.
I'm agree with preparing TAE+BrEt stock. It works very good for both gels and running buffer and never have had problems with heating. For storing we use an ambar recipient. BrEt is at same concentration that Montana81 says.
Regards!
We also add EtBr under a fume hood after microwaving and cooling down the gel a little bit. But: Some people tell that EtBr evaporates, some say it does not. ??? My guess is that it is in the steam/aerosol when heating it.
On the fact sheet nothing is mentioned about that. But since it is a quite heavy organic molecule (394 g/mole) and solid (if not in solution), it should have no gas phase (or it is destroyed during heating, the melting point is at 260°C).
Is this correct or has anybody ideas??
EtBr is mutagen. It's harmful if swallowed of inhalated.
So, it's better to work under a hood, and not to boil EtBr.
It's not so difficult to add EtBr only when the gel is melted, and this could save your life and the one of your colleagues.
so, why to take the risk? tell me.