Ethidium bromide gel - (May/09/2007 )
i agree 100% with beccaf22. really you dont need to be worried about the black part.
we never put EdBro in TAE when we use it as runing buffer
but we put EdBr in TAE when we store gel in it
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
This always happens (to me at least), especially if I run gel for long time (1h or so) and I want the lower band, sometimes is a bit hard to see the smaller bands (<200bp). Don't worry. beccaf22 is right. When I need the lower band, I either stain the gel later or put EtBr into the positive side chamber like Nabin said. About the same or less concentration as with the amount you put in gel. If you don't want the small bands, forget it.
We never put EtBr in the running buffer.
As far as I am aware, however, I don't think that the lack of EtBr in your gel will affect your ability to see bands, in actual fact I think that it improves it. The EtBr will be incorporated into you DNA/PCR product so it is not important whether or not there is any left in your gel. We will often run a gel (particularly for RFLP) until all the EtBr has run out of the gel, so there is no background illumination and the bands appear more clearly.
Many thanks for all the replies! The bands I am looking at are 150-300bp, but I have been able to see them fine so I guess this isn't something that I need to worry about then, thanks again!
You have too much ethidium bromide in the gel. See how the top part of the gel is lighter than the lower part of the gel (ignoring the bands)? That's the background ethidium bromide which has migrated upwards. You shouldn't actually see any background EtBr - it should all look like the lower part. You said yourself that you can see your bands just fine even when they're lower down. This is because EtBr doesn't migrate so much when it's interacalated with the DNA.
Yes, Zouden I think you are right, yesterday I reduced the amount of EtBr in my gel and it looked far better. Thanks for all the help!