Ethidium bromide gel - (May/09/2007 )
This is going to seem like a really stupid question but here goes anyway. In my last lab we used to stain our DNA gels after running them (boss insisted, we had no choice). Anyway now that I have moved lab I don't want to waste my time staining and destaining so I decided to include the EtBr in the gel. However when I go to look at the gel under the UV, the upper half of the gel seems to have far more EtBr than the lower half! I give the gel a good swirl before pouring so I really don't think it could be that it's not mixed properly. I have tried putting in more EtBr to the gel also but it hasn't made much difference.
If anyone has any suggestions I would really apprecicte it, thanks, auldmok
how much EtBr dp ypu useµ? i use 1µl of 10mg/ml stock solution for 100ml agarose gel and it's fine
2nd is that EtBr moves at opposite sense as DNA so downer part tend to de-concentrate quicker as upper one.
what do you mean when you have less EtBr in the lower half? How do you check that out?
Normally, at the lower half of the gel, the EtBr is usually little as the band size is significantly less in comparison to the upper level isnt it?
If mixing is not the case, I am guessing it might be your buffer or during electrophoresis.
Don't you need to put Ed Bro in TAE when running the gel? Do you do that?
i've never done that
Yea, I dont think you have to put EtBr into the TAE buffer. Just the agarose gel is enough.
We use 4ul of 10mg/ml EtBr stock per 100ml agarose. Check pH of your buffer.....also quality of agarose.
I've never even heard of putting the EtBr in the buffer instead of/along with the agarose . . . wouldn't that make a huge mess to clean when it's time to discard buffer? Putting it in the gel only probably makes a lot less peripheral contamination, I should think.
When you run a gel containing ethidium bromide, the ethidium is attracted to the negative pole while the DNA moves toward the positive pole. This causes the Ethidium to make like a line in the gel where the top part is stained and the bottom is not because the ethidium is pulled through and out of the gel.... This may or may not affect your ability to see the bands, one way to keep this from happening (and yes it is a nightmare to clean up) is to add ethidium to the TAE buffer at the same concentration as you have in the gel. This way the ethidium goes into the gel at the same rate it is pulled thru the gel to the negative electrode and you don't see the line. We never did that (again royal pain to clean up all that EtBr contaminated buffer) instead we ran the gels for amounts of time that kept the "ethidum line" below the bottom of the bands we were detecting, all my gels had a line at the bottom that looked like the stain had gone out of the bottom of the gel but you just ignore that. Adding more EtBr does not solve this problem, but makes it worse... umm I hope this is clear post again if you have any questions and good luck!!
I have not run gels that many times to be able to give any suggestion and only for one little part of experiment I need to do that. When I was taught by my senior, she told me to put EtBr also at the bottom of the electrophoresis tank so that it moves on the opposite direction and the gel is uniform.
Now, I understand that may not be necessary if I can do like what beccaf22 has suggested. But, we put extra EtBr or not, the buffer should still be disposed with all the care needed.