Agarose gel electrophoresis troubleshooting - (May/01/2009 )

HomeBrew on Jun 12 2009, 10:36 PM said:

I am always adding EtBr directly to the gel and always get beautiful gels; never had any problems with visualising my products (long and short ones). Worst case was that the gel had run dark, but the DNA was still brightly shining at me.

Although late, but another suggestion for the problem: have you checked the power supply???? I have faced a similar problem once: The loading dye was slowly diffunding into the gel and was fading away....so I found out that our power supply was broken.

same here.. never had problems with staining the gel right before pouring, even with 70kb size fragments.

how long do you run your gels?
it might work better if you don't run them for too long.

could it be the problem you are seeing is actually high background (ie by adding too much etbr or by having a gel that is not clean (ie bad brand, dust in the agarose, re-used a gel too often)?

i have made the experience that many people actually add too much etbr to their gels, increasing the background and lowering the s/n ratio.

warsel on Jun 22 2009, 09:59 PM said:

Hi warsel, can i check to confirm that you stained your agarose gel before casting, and while running such large fragments, you don't have problem with the separation of the large bands? i'm asking this because I'm looking to buy some large dna ladder and its recommended that i do post gel staining as doing pre-staining might affect separation of the larger bands of the ladder. Also, what the % of gel you use and conditions you run your gel for? Thanks in advance.

just add a little etbr in the tank buffer and the bfr running the gel the bands would not fade

Ethidium bromide is light sensitive. Make sure that stock of it was kept in dark. If it's exposed to light long time, you will get very week signal.
http://www.bioprotocols.info

Maybe stupid insinuation: i hope the UV-illuminator is still working? UV-bulbs age and can be the reason for seeing faint bands?

perhaps the EB is unavailable and out of date so please try a new one

Lazinase on Fri May 8 06:08:53 2009 said:

if so it sounds like there is something wrong with your etbr. I have never done this before though, do you think its possible to mix your sample with a little amount of etbr and check it under the UV.

chromatin on Thu Jul 29 01:39:12 2010 said:

That's what I was about to suggest, try a different EtBr stock.
You can see the EtBr running in the opposite durection than the sample, so the bottom of the gel is much less bright. Are you running your samples long and out of the EtBr? 2% gel is pretty thick, I usually run 1%, otherwise the samples run slowly and the EtBr may run out of the gel by the time you're done. Good luck

kdw on Fri May 1 21:58:33 2009 said:

Do you mean after they prepare the TBE solution, they add EtBr inside directly, and you just add in your agarose powder, microwave and mold it?
I thought you should only add EtBr after microwave and before you mold it? This is how I do my gel.