more ethidium bromide, brighter band? - (Sep/17/2006 )
Hi,
I have about 30ng of DNA product. I am afraid that I can't see the band clearly when I run on a gel. I usually add 1ul of EtBr to 40ul of agarose. I wonder whether I can add more EtBr so that I see a brighter band. Is it possible?
Thank you.
We add 1ul to 30 ml gel. I wouldn't add more because you would increase the background fluorescence. You should be careful with the run because your product is very small and the EtBr runs to the opposite direction as the DNA. I would run the gel not for too long, If you have problems because you realize that the EtBr is not present anymore where your piece should be, you can stain with EtBr after the electrophoresis
I have about 30ng of DNA product. I am afraid that I can't see the band clearly when I run on a gel. I usually add 1ul of EtBr to 40ul of agarose. I wonder whether I can add more EtBr so that I see a brighter band. Is it possible?
Thank you.
You would get better result by staining the gel after the electrophoresis run. And a short 5min destaining with water if the background is too high.
In your case adding more EtBr would increase band brightness... mainly because most of your EtBr is being pulled out our gel during the electrophoresis.
But personally I would not recommend using more EtBr.. the stuff isn't good. I am sure 20 years from now, we'll say "Back in my day we didn't bother about all this safety nonsence. We had EtBr all over the place."
My supervisor say that about radioisotopes.
actually, the brightness of the bands is depended on the DNA concentration. SO even u add more EtBr which your DNA amount is very little, then you still cannot get the brighter band
True. But in this situation EtBr was added during the eletrophoresis run. The bands are usually fainter under such staining conditions. Not noticible for most quantities of DNA, but can make a difference when looking a faint bands.
For maximum brightness, ie maximum EtBr uptake, staining the gel after the run.
In anycase the amount of EtBr added here is nowhere near the amount used to make the EtBr bath that my lab uses.
I know it is bad.. but it is very fast.... 5min to 10min and the gel is stained. Fish out the gel like french fries and image the thing.