Agrose gel - (Oct/30/2008 )
I make lot og agrose gel. Can I make a stock solution of agrose gel and usedit again and again.
At which step ethedium bromide have to put it in stock solution.
At which step ethedium bromide have to put it in stock solution.
For me it worked fine to prepare a stock and keep it for up to 48h at 70°C, take out the amount you need THEN add EtBr and then pour it. But it also works fine if you prepare a stock and melt it every time you need it, take out the amount you need THEN add EtBr etc.
But I would only use this for routine-gels and not for crucial gels, as the agarose-gel changes its properties when kept at 70°C for a long time or is re-melted again and again.
When we're done using an agarose gel, we just break it into pieces and keep it in a flask with a seal over the top. the next time you want to use it, just melt it again. It does not seem to affect the ethidium bromide. I will reuse the agarose until it becomes really discolored from loading dye or the brightness of the bands seems to have gone down too much (you can tell this from your DNA ladder). I would not recommend re-using the agarose if you are gel isolating something.
You can also reuse the same gel many times without remelting. Whatever ethidium bromide was in the gel at the beginning will, of course, eventually run off, but then you can just soak the gel for 10-15 minutes in an EtBr solution after your run.
@smu2 & wbla335: We used to re-melt and re-run gels too, but I was always getting the feeling that the bands are getting more and more diffuse and do not segregate well any more. Is this just paranoia or are you experiencing similar pattern?
We stoped re-melting the gels as we do not have a fume hood for the microwave and EtBr can be dissolved in the steam from the re-melted gels....
We stoped re-melting the gels as we do not have a fume hood for the microwave and EtBr can be dissolved in the steam from the re-melted gels....
I usually do not heat the gels so much that I notice a lot of steam coming off of them - for about 40 ml of gel I heat for 45 seconds. As for the bands getting more diffuse, I usually reuse my gel no more than 4-5 times so maybe that has something to do with it. It could also depend on what type of agar or what percentage you are using. I usually use seakemp or nusieve. If I notice that the gel has dried up quite a bit then I will add a little more TBE to it before I pour it - could be that your gel is getting more concentrated and this is why the bands are not segregating.
Another note to those who want to save time - I was in a lab once where they used to pour large agarose gels (about the size of 6 minigels- and then put them in the fridge. They didn't add EtBr to the gel. Then the whole lab shared the gel- just cutting off the portion that they used with a scalpel- and then did the EtBr soak. It was a little bit messier, but it did save time when you were in a hurry to see things.
I would prefer to keep the melt agarose gel in 70oC oven. Too many times of heating will cause the agarose gel becomes more concentrated, and end up not to be the concentration required. Also, as some of you mentioned, the DNA is not separated well after too frequent heating. That's a waste of the time spent on an experiment.